Treatment of post-bariatric hypoglycemia with GLP-1 antagonists

ABSTRACT

Treatment of hyperinsulinemic hypoglycemia comprises administration of an effective amount of a glucagon-like peptide-1 receptor antagonist (GLP1RA) alone or in combination with an amylinomimetic agent or any anti-gastric emptying agent. Patients suffering from hyperinsulinemic hypoglycemia after bariatric surgery experience particular benefit, as there is no current method effective for their treatment. Prevention or reduction of acute adverse effects of postprandial hypoglycemia, such as palpitations, tremor, weakness, sweating, confusion, fatigue, blurred vision, seizures, or loss of consciousness, and prevention of chronic adverse effects of hyperinsulinemic hypoglycemia, such as cognitive impairment, can be achieved by treatment with GLP1RA.

This application is a continuation of U.S. patent application Ser. No.15/576,646, filed Nov. 22, 2017, which is the National Stage ofInternational Application No. PCT/US2016/033836, filed May 23, 2016,which claims priority to U.S. Provisional Application No. 62/329,850,filed Apr. 29, 2016, U.S. Provisional Application No. 62/254,175, filedNov. 11, 2015, and U.S. Provisional Application No. 62/165,743, filed onMay 22, 2015, the contents of each of which are incorporated byreference herein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under contract TR001085awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention provides methods and compositions for thetreatment of hypoglycemia, particularly post-bariatric hyperinsulinemia,and more generally hyperinsulinemic hypoglycemia of any origin, and theprevention of associated acute symptoms and chronic outcomes in which aglucagon-like peptide-1 receptor antagonist (GLP1RA) is formulated foradministration and administered in a therapeutically effective dose,often by route of subcutaneous injection, either alone or in combinationwith an amylinomimetic or other anti-gastric emptying agent. In variousembodiments, the patient receiving therapy has had bariatric surgery, ametabolic procedure, or a gastrointestinal surgery, and thehyperinsulinemic hypoglycemia (“hyperinsulinemia” or more generallyhypoglycemia) suffered by the patient is believed by his or herphysician to be due to complications of that surgery or some otherprocedure or condition likely to result in similar disease pathology.The invention therefore relates to the fields of biology, chemistry,medicinal chemistry, medicine, molecular biology, and pharmacology.

BACKGROUND OF THE INVENTION

Roux-en-Y gastric bypass (RYGB), widely performed for medicallycomplicated obesity, cures type 2 diabetes in 85% of cases. Thephysiologic mechanisms mediating diabetes resolution is controversial,but the reduction in glucose excursions prior to weight loss has led topostulates that the incretin hormone, glucagon-like peptide-1 (GLP-1),may play an important role. GLP-1 stimulates the secretion of insulin bypancreatic beta cells and is responsible for the “incretin” effect:incretin hormones enhance the glucose-dependent secretion of insulin,such that pancreatic beta cells will secrete more insulin after an oralglucose load than after an isoglycemic IV glucose load. Enhancedsecretion of GLP-1 after RYGB, and a resultant elevation in insulinsecretion, may play a primary role in the resolution of diabetes afterRYGB. Indeed GLP-1 analogs have been developed to treat diabetes.

However, as the use of bariatric surgical procedures continues toincrease worldwide, a severe complication—hyperinsulinemichypoglycemia—is increasingly reported. Present in 1-6% of RYGB patients,this disorder leads to severe symptomatic hypoglycemia that plaguespatients often multiple times daily, with glucose concentrations lowenough (20-40 mg/dL) to cause seizures, altered mental status, loss ofconsciousness, cognitive dysfunction, disability, and death. Quality oflife is severely diminished, and many patients cannot care forthemselves or others, work, drive, or be left alone. There is noeffective treatment and severe cases have been managed with near-totalto total pancreatectomy, which results in insulin-dependent diabetes andis associated with a 6% surgical mortality risk.

Given the severity of this chronic disorder with unmet clinical need, aneffective therapeutic treatment is urgently needed. It would thus beuseful to provide a method for the treatment of hyperinsulinemichypoglycemia post bariatric surgery and prevention of its acute symptomsand chronic outcomes, and a pharmaceutical composition for suchtherapeutic.

SUMMARY OF THE INVENTION

While the present invention provides a variety of methods and materialsfor treatment and prevention of hyperinsulinemic hypoglycemia, oneaspect of this invention relates to the pharmaceutical compositions andmethods involving subcutaneous delivery of a GLP-1 receptor antagonist(“GLP-1RA”) in doses therapeutically effective for this indication. Insome instances, exendin(9-39) is delivered subcutaneously as a GLP-1RAfor this indication. In many embodiments, subcutaneous GLP-1RAformulations, including formulations of exendin(9-39), are provided asimmediate release preparations or as extended/slow release preparations,and are conveniently packaged, for example, in the form of thedual-chamber pen device provided by the invention, or an alternate vialand syringe combination provided by the invention for patients toself-administer, or their care providers to administer, therapeuticallyeffective amounts of the GLP-1RA.

For exendin(9-39) and GLP-1RAs of similar activity and in similarformulations to achieve similar pharmacokinetics as described herein,therapeutically effective doses range from 2-100 mg. In someembodiments, exendin(9-39) is formulated and administered in aninjectable pen device or via a vial/syringe combination that may bepre-programmed or marked to deliver a fixed dosage amount ranging from2-100 mg, 10-75 mg, 20-50 mg, 20-40 mg, 2-10 mg, 2.5-10 mg, or 2.5-7.5mg, depending upon the needs and physical attributes of the patient. Insome embodiments, adult patients (60-100 kg or more) will receivetherapeutic benefit from a single dose in the range of 10-75 mg, withsome achieving full therapeutic effect with 20-40 mg. Each dose will beadministered in a total volume ranging from 0.25-2 ml injectate. In someembodiments, adult patients (and some adolescents and minors) willbenefit from doses as low as 5 mg or from 2.5 mg to 5 mg, and typicaldoses will often range in the 2.5 mg to 10 mg, 2.5 mg to 7.5 mg, or 5 mgto 30 mg dose. As noted, most patients will self-administer this dose atleast once a day, and often twice a day. Some patients will administerthis dose with each meal, or before a particular meal, including, forexample a certain time, e.g., 15 minutes to two hours, e.g. one hour,before a meal, or a certain time after a meal.

In one embodiment, the patient self-administers a dose of exendin(9-39)in the range of 5 mg to 30 mg or 2.5 mg to 10 mg in an immediate releaseor extended release formulation. In one embodiment, this dose isadministered BID using an immediate release formulation, with the firstdose in the morning, typically before the first meal, e.g. at least 60minutes before, and the second about twelve hours later. In anotherembodiment, this dose is administered qD in an extended releaseformulation that is dosed either in the morning, as above, or in theevening, e.g., before or after the last meal of the day, including, e.g.before retiring. For these various embodiments, the invention providesimmediate release formulations of exendin(9-39) and formulations similarto that of Byetta® (Amylin; AstraZeneca), a subcutaneously administeredform of exenatide. In other embodiments, the formulation is an extendedrelease formulation of the invention suitable for once-a-weeksubcutaneous administration that is similar to the Bydureon® (Amylin;AstraZeneca) formulation that provides for once-a-week dosing ofexenatide. In other embodiments, the formulation is an extended releaseformulation of the invention suitable for once-a-day subcutaneousadministration that is similar to the Victoza® (Novo Nordisk)formulation that provides for a once-a-day dosing of the GLP-1 agonistliraglutide.

In various embodiments, the invention provides immediate release anddelayed release formulations of exendin(9-39) and methods for theirdelivery by subcutaneous administration to post-bariatric surgerypatients suffering from hyperinsulinemia (hypoglycemia). In oneembodiment of the invention, the patient self-administers (or is wearinga device programmed to administer) the exendin(9-39) in a dose rangingfrom 4-40 mg, although many patients will receive therapeutic benefit bya dose ranging from 10-25 mg. Dosing will typically be qD or BID, withqD administration typically utilizing a delayed or extended releaseformulation of the invention. The first dose may be administered in theevening, such that it provides protection during breakfast the followingday, with subsequent doses following the next evening and each eveningthereafter for qD administration or the following morning and abouttwelve hours later for BID administration. In one embodiment, atherapeutically effective amount of exendin(9-39) is administered viasubcutaneous injection to provide treatment benefit similar to thatachieved by continuous IV infusion of exendin(9-39), which effectivelyreverses postprandial hyperinsulinemic hypoglycemia and associatedsymptoms. In one embodiment, a patient receives an intravenous (IV)infusion of a GLP1A, followed by administration of a therapeuticallyeffective dose of a GLP1A by subcutaneous injection, or by another routeof administration described herein. While continuous IV infusion can beused in the in-patient hospital setting for chronic or acute severehypoglycemia, this may not be a practical or realistic outpatient methodof treatment. The pharmacokinetic properties of intravenouslyadministered exendin(9-39) (for example, a half-life (T1/2) of 33.5minutes, a volume of distribution (V_(d)) of 111 ml/kg, and a drugclearance (CL) of 2.3 ml·kg⁻¹·min⁻¹), however, are such that one ofordinary skill in the art would conclude that continuous IV infusion ofexendin(9-39) would be required in order to achieve a therapeuticresponse.

In accordance with the methods of the invention, however, other routesand forms of administration may be employed with significant benefitboth to patient and physician. For example, a single IV bolus dose ofthe exendin(9-39) administered as described herein can effectivelyprevent hypoglycemia despite its half-life of 33.5 minutes (see FIGS.4A-D and Example 2). While this method provides some treatment benefit,if the administration is not timed to coincide precisely with the GLP-1peak plasma concentrations, IV bolus administration can fail to preventhypoglycemia.

More importantly, for most patients, subcutaneous injection ofexendin(9-39) as described herein can provide even more efficaciousresults. The present invention can be practiced with an immediaterelease formulation of exendin(9-39) that, when used in accordance withthe invention, will effectively prevent hypoglycemia in most patients.Furthermore, for this and other formulations, there is no requirement,at least for the vast majority of patients, to time the administrationto peak GLP-1 plasma concentration or any other biomarker.

Many patients, however, will achieve good control of their glucose andmuch prefer the convenience of one of the extended release formulationsof the invention for subcutaneous injection, e.g., a once-a-weekformulation, and some patients can benefit from the oral, inhaled, ornasal formulations provided.

In addition, in some embodiments of the method, some patients will enjoyenhanced therapeutic benefit from the co-administration of anamylinomimetic or any agent that has the effect of delaying gastricemptying. Consequently, the present invention also provides forcombination therapies in which a GLP-1A, such as exendin(9-39), iscoadministered with an agent that delays gastric emptying, such as anamylin peptide (e.g. Symlin), to improve the treatment ofhyperinsulinemic hypoglycemia, or with another agent that delays gastricemptying, such as an aluminum hydroxide antacid, any H2 ReceptorAntagonist (e.g. Ranitidine, Cimetidine, or Famotidine), or any ProtonPump Inhibitor, (e.g. Omeprazole, Lansoprazole, or Pantoprazole), or anycombination of any of the foregoing. The pharmaceutical compositions ofthe invention have use for treatment and prevention of hyperinsulinemichypoglycemia and its associated symptoms and outcomes in patients withhyperinsulinemic hypoglycemia post bariatric surgery and postgastrointestinal surgery. In addition, because hypoglycemia can beaverted in a GLP-1-dependent manner, the compositions of the inventionare also useful as an immediate antidote for any over-exposure to GLP-1or similarly acting GLP-1 analogue, such as in instances of postprandialhypoglycemia that do not occur in the postbariatric or postgastrointestinal surgery setting, and in instances of acute overdosingof a GLP-1 agonist. GLP-1 agonist drugs are well known in the art andinclude exenatide, liraglutide, lixisenatide, albiglutide, anddulaglutide.

In these and other embodiments, the invention provides for theprevention and treatment of associated acute and chronic symptoms andoutcomes in susceptible patients. Treatment in accordance with theinvention of patients in need of therapy will improve patient quality oflife both in the short- and long-term, will reduce overall patientmorbidity, and may prevent premature death and/or extendlife-expectancy. The present invention is believed to be the first safeand effective treatment for hyperinsulinemic hypoglycemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B. Average plasma glucose response (FIG. 1A) andaverage insulin response (FIG. 1B) to a 75 gram oral glucose tolerancetest (OGTT) in subjects with hyperinsulinemic hypoglycemia during arandomized blinded cross-over study in which a primed continuousintravenous (IV) infusion of exendin(9-39) (at 500 pmol/kg/min over 180minutes) or placebo (normal saline) was administered, as described inExample 1. Solid line: placebo infusion; dashed line: exendin(9-39)infusion.

FIG. 2A, FIG. 2B and FIG. 2C. Average plasma GLP-1 response (FIG. 2A),average GIP response (FIG. 2B), and average glucagon response (FIG. 2C)to a 75 gram OGTT in subjects with hyperinsulinemic hypoglycemiareceiving primed continuous IV infusion of exendin(9-39) of 500pmol/kg/min over 180 minutes versus placebo (normal saline) infusion, asdescribed in Example 1. Solid line: placebo infusion; dashed line:exendin(9-39) infusion.

FIG. 3. Individual and average symptomatic responses to a 75 gram OGTTin 8 patients with hyperinsulinemic hypoglycemia receiving a primedcontinuous IV infusion of exendin(9-39) versus placebo, as described inExample 1. Overall Symptom Score, Glucose Rise, and Glucose Fall Scoresare presented. Continuous IV infusion of exendin(9-39) at 500pmol/kg/min over 180 minutes substantially improved symptoms ofhypoglycemia, as demonstrated by the reduced Overall Symptom and GlucoseFall scores.

FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D. Plasma glucose responses to IVbolus doses of 7,500 pmol/kg of exendin(9-39) administered to a subjectwith hyperinsulinemic hypoglycemia at different time points relative tothe timing of administration of glucola in an OGTT. Specifically, theexendin(9-39) IV bolus was administered at: T=0 minutes (FIG. 4A), T=20minutes (FIG. 4B), or T=50 minutes (FIG. 4C). For FIGS. 4A-4C, glucoselevels measured at specific timepoints as described in Example 2 areshown by a solid line. For FIGS. 4A-4D, the projected exendin(9-39) PKcurve after administration of the IV bolus of 7,500 pmol/kg is shown bya dotted line. Dosing at 0 minutes (FIG. 4A) or 20 minutes (FIG. 4B) didnot prevent hypoglycemia, whereas dosing at 50 minutes (FIG. 4C) didprevent hypoglycemia. The GLP-1 peak for the subject in FIG. 4C occurredat 60 minutes, suggesting that timing the administration of the IV bolusto the GLP-1 plasma peak was necessary to prevent hypoglycemia.

FIG. 5A and FIG. 5B. In FIG. 5A, average plasma exendin(9-39)concentrations for 8 human subjects administered a continuousexendin(9-39) IV infusion at a rate of 500 pmol/kg/min over 180 minutesare plotted (black line), wherein the projected exendin(9-39)pharmacokinetic response to a single IV bolus of 7,500 pmol/kgexendin(9-39) administered at T-30 (blue line) was extrapolated based onthe known half-life of intravenously administered exendin(9-39). In FIG.5B, a single IV bolus of 7,500 pmol/kg exendin(9-39) or a singlesubcutaneous injection of 7,500 pmol/kg exendin(9-39) was administeredto a subject, wherein plasma exendin(9-39) concentrations were measuredby liquid chromatographymass spectrometry (LCMS), and wherein the Cmaxthat was observed in subcutaneous administration of exendin(9-39) wassignificantly lower than the Cmax observed in intravenous administrationof exendin(9-39).

FIG. 6. Average plasma glucose levels during a 75 gram OGTT for subjectsadministered a subcutaneous dose of exendin(9-39) as compared tobaseline. Four subjects received one subcutaneous injection of 35,700pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg (approximately 10, 20, or 30mg, respectively, based on an 80 kg patient) in a volume of 0.7 mlnormal saline, and four subjects received two or more 0.7 ml injectionsof 35,700 pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg in order tomaintain an injectate concentration of 15 mg/ml or less. The averageplasma glucose nadir for all 8 subjects during subcutaneous injection ofexendin(9-39) was 78 mg/dL vs. <50 mg/dL during a baseline oral glucosetolerance test, demonstrating that subcutaneous injection of a singledose about 10-30 mg exendin(9-39) was able to effectively reversehyperinsulinemic hypoglycemia.

FIG. 7A and FIG. 7B. FIGS. 7A and 7B demonstrate plasma exendin(9-39)concentrations following subcutaneous injection of exendin(9-39). InFIG. 7A, three subjects received a single subcutaneous injection ofapproximately 10, 20, or 30 mg of exendin(9-39) in a volume of 0.7 ml(5×, 10×, or 15× doses, respectively). In FIG. 7B, five subjectsreceived doses of approximately 2, 10, 20, or 30 mg (1×, 5×, 10, or 15×,respectively), with each dose administered at a concentration of 15mg/ml or less; higher doses were administered via more than oneinjection so as to maintain a relatively dilute concentration.

FIG. 8. Percent increase in plasma glucose nadir concentrations werecalculated for the subcutaneously administered doses of exendin(9-39)relative to baseline. A correlation was observed between higher percentincreases in plasma glucose nadir concentrations and increasing peakplasma exendin(9-39) concentrations (C_(max)).

FIG. 9. Study design for 3-day Multi-Ascending Dose Trial to assess thesafety, tolerability, efficacy, and pharmacokinetic profile of BIDexendin(9-39) administered subcutaneously over 3 days to patients withpost-bariatric hyperinsulinemic hypoglycemia.

FIG. 10. Exendin(9-39) plasma concentrations on Day 3 after 5 doses asdescribed in Example 4.

FIG. 11. Diminished novelty preference scores on the Visual PairedComparison (VPC) Test in patients with hyperinsulinemic hypoglycemia ascompared to insulin sensitive healthy controls predicts mild cognitiveimpairement indicative of cumulative hyperinsulinemic hypoglycemiaeffects. As described in Example 6, patients with hyperinsulinemichypoglycemia exhibited early signs of amnestic mild cognitive impairment(aMCI) as measured by the VPC test in comparison to healthy controls,and therein may benefit from GLP1A for prevention of further cognitivedecline.

BRIEF DESCRIPTION OF THE TABLES

Table 1: Metabolic responses to a 75 gram oral glucose tolerance test(OGTT) during primed continuous IV infusion of exendin(9-39) in eightpost-RYGB patients with hyperinsulinemic hypoglycemia (HH). Metabolicresponses of eight BMI, age, and sex matched non-surgical controls arepresented for comparison. AUC values were calculated by the trapezoidalrule utilizing the last value carried forward to account for prematurelydiscontinued OGTTs in cases of hypoglycemia, which occurred solelyduring placebo infusion.

Table 2: Mean plasma GLP-1, GIP and glucagon response to a 75 gram oralglucose tolerance test (OGTT) in eight patients with hyperinsulinemichypoglycemia (HH) during a primed continuous IV infusion ofexendin(9-39) of 500 pmol/kg/min over 180 minutes vs. during placebo(normal saline) infusion.

Table 3: Subject metabolic and symptomatic response to a singlesubcutaneous (SC) injection of 10-30 mg of exendin(9-39), denoted as SCEx(9-39), continuous IV infusion of exendin(9-39 (IV Ex(9-39)), orplacebo during a 75 gram OGTT. This table demonstrates that clinicalefficacy during this SAD subcutaneous injection study was comparable tothat achieved during continuous IV infusion of exendin(9-39), asmeasured by the plasma glucose nadir, AUC glucose, and the Symptom FallScore.

Table 4: PK/PD response to increasing doses/increasing concentrations.As described in Example 3, subjects 2-5 each received a subcutaneousinjection of exendin(9-39) in doses ranging from 37,500-112,500 pmol/kg(approximately 10-30 mg) each in a volume of 0.7 ml, resulting in doseconcentrations of approximately 15-40 mg/ml. Shown here are subjectPK/PD responses to each dose. Injectate concentrations of approximately15 mg/ml resulted in the greatest pharmacodynamic response, as definedby nadir postprandial glucose and AUC glucose, and greatestpharmacokinetic response, as defined by Cmax and DN Cmax. Thus arelatively dilute dose may be preferred for BID dosing, and a moreconcentrated formulation may be preferred for less frequent dosing or amore sustained exposure. The 75,000 pmol/kg dose (17 mg) with aconcentration of about 24 mg/ml resulted in a favorable sustained/slowrelease pharmacokinetic response, with a half-life of 9.14 hours, and aCmax that was 70 or more ng/ml. Thus a relatively concentrated dose maybe used advantageously for qD or BID dosing not tied to meals.

Table 5: PK/PD response to increasing dose with constant injectateconcentration. As described in Example 3, four subjects receivedsubcutaneous injections of 37,500-112,500 pmol/kg exendin(9-39) inequivalent concentrations (approximately 13-16 mg/ml), as thisconcentration was found to result in a favorable immediate releaseformulation of the invention. Results shown demonstrate an increasinglyfavorable PK response with increasing dose, as defined by Cmax andT_(1/2).

Table 6: PK/PD response in four subjects dosed with varying doses ofsubcutaneously administered BID exendin(9-39) in a 3-day clinical trialas described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION 1. Introduction

Post-bariatric hyperinsulinemic hypoglycemia is a disorder that ischaracterized by low blood sugar and elevated insulin levels 1-3 hoursafter meals. The disorder manifests as neuroglycopenic symptoms (such asconfusion, loss of focus, fatigue, ataxia, paralysis, seizures, or lossof consciousness), vasomotor symptoms (such as sweating and shakiness),and/or adrengeric symptoms (such as heart palpitations). Although thepathogenesis of post-bariatric hyperinsulinemic hypoglycemia is notentirely understood, several mechanisms have been proposed, includingincreased section of insulinotropic incretin gut hormones from thehindgut, expansion in β-cell mass, enhanced β-cell sensitivity,increased insulin sensitivity, decreased insulin clearance, reducedability to mount a counterregulatory glucagon response, and absence of aprodiabetogenic/decretin foregut factor.

As described in Example 1, administration of a Glucagon-like Peptide-1(GLP-1) receptor antagonist, exendin(9-39), by primed continuousintravenous (IV) infusion of exendin(9-39) effectively reversedhyperinsulinemic hypoglycemia and associated symptoms in patients havinghyperinsulinemic hypoglycemia. In this trial patients received a totaldose about 23-36 mg exendin(9-39), with the drug quantity varying withpatient weight. (Also see Salehi et al., 2014, “Blockade ofGlucagon-like Peptide 1 Receptor Corrects Postprandial HypoglycemiaAfter Gastric Bypass,” Gastroenterology 146:669-680.) However, differentpharmacokinetic profiles would be expected, and are observed, for asubcutaneously administered GLP-1 antagonist such as exendin(9-39) ascompared to intravenous administration. See Example 3. Accordingly, oneof ordinary skill in the art would have expected that a significantlyhigher dose of a GLP-1 antagonist such as exendin(9-39) would be neededfor subcutaneous administration, as compared to intravenousadministration, in order for exendin(9-39), to be effective in reversinghyperinsulinemic hypoglycemia.

It has been surprisingly shown that subcutaneous administration ofexendin(9-39) can effectively prevent hypoglycemia in patients havingpost-bariatric hyperinsulinemic hypoglycemia, even at doses lower thanthe doses administered in continuous primed IV infusion as described inExample 1. See, Example 3 and Example 4. Thus, in one aspect, thepresent invention relates to pharmaceutical compositions and methods forsubcutaneously administering a GLP-1 antagonist at a therapeuticallyeffective dose for the treatment and prevention of hyperinsulinemichypoglycemia.

It will be recognized by physicians and pharmacologists that the presentinvention represents a significant advance in the field of surgicalintervention for weight loss and/or metabolic control. This isespecially important, because those post-bariatric patients currentlysuffering hypoglycemic excursions have no effective therapy and aresometimes critically ill. The intractable nature of the problem has beenhighlighted by those patients with disease so debilitating they reversedthe surgery, or underwent other highly morbid procedures, such aspartial pancreatectomy, only to learn the condition persists. Thepresent invention is a significant advance for this reason alone: thosepatients now have a therapeutic intervention that can largely protectthem should they suffer from post-bariatric hyperinsulinemia.

2. Definitions

The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention pertains. In some cases, terms with commonlyunderstood meanings are defined herein for clarity and/or for readyreference, and the inclusion of such definitions herein should not beconstrued as representing a substantial difference over the definitionof the term as generally understood in the art.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Alltechnical and patent publications cited herein are incorporated hereinby reference in their entirety.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1 or 1.0, as appropriate. It is tobe understood, although not always explicitly stated, that all numericaldesignations are preceded by the term “about.” References to rangesinclude the end-points unless indicated otherwise. For example,administration of a dose of a GLP-1 antagonist in the range 10-75 mgincludes administration of 10 mg or 75 mg.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a compound” includes a plurality of compounds.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of” shall mean excluding other elements thatwould materially affect the basic and novel characteristics of theclaimed invention. “Consisting of” shall mean excluding any element,step, or ingredient not specified in the claim. Embodiments defined byeach of these transition terms are within the scope of this invention.

The terms “administer,” “administering,” and “administration,” as usedherein, refer to introducing a compound (e.g., exendin(9-39) orcomposition into a subject, such as a human. As used herein, the termsencompass both direct administration (e.g., administration to a subjectby a medical professional or other caregiver, or by self-administration,or by programming an automatic device to deliver a GLP-1 antagonist on aschedule) and indirect administration (e.g., the act of prescribing acompound or composition to a subject).

The terms “treatment,” “treating,” and “treat,” as used herein inreference to administering a GLP-1 antagonist to treat hyperinsulinemichypoglycemia covers any treatment of a disease in a human subject, andincludes: (a) reducing the risk, frequency or severity of hypoglycemicepisodes in patients with a history of hyperinsulinemic hypoglycemia,(b) reducing the risk of occurrence of hypoglycemia in a subjectdetermined to be predisposed to the disease, such as a person who hasreceived post-bariatric surgery, but not yet diagnosed as having thedisease, (c) impeding the development of the disease; and/or (d)relieving the disease, i.e., causing regression of the disease and/orrelieving one or more disease symptoms.

As used herein, the term “injectate” refers to a GLP1A-containing (e.g.,an exendin(9-39)-containing) composition that is delivered to a patientat a morning or evening administration. A morning or evening injectateis typically administered as a single injection (e.g., injection of a0.7 ml volume). However an injectate can be delivered using more thanone (e.g., two) injections, as may be done when the injectate volume isgreater than comfortably tolerated as a single injection.

As used herein, “/kg” (e.g., 7,500 pmol/kg”) means “per kilogram patientbody weight.”

3. Methods and Compositions for the Treatment of HyperinsulinemicHypoglycemia

In one aspect, the present invention provides methods and compositionsfor the treatment of hyperinsulinemic hypoglycemia by administration ofa therapeutically effective dose of a GLP-1 antagonist.

3.1 GLP-1 Antagonists

In one aspect, the present invention provides pharmaceuticalcompositions and methods involving administration of a GLP-1 receptorantagonist (“GLP-1RA”), also referred to as GLP-1 antagonist (“GLP1A”),and so used interchangeably herein.

In various embodiments, the GLP1A is exendin(9-39). As used herein, theterm “exendin(9-39)” or “Ex(9-39)” or “Ex9” refers to a 31 amino acidpeptide with an empirical formula of C₁₄₉H₂₃₄N₄₀O₄₇S and a molecularweight of 3369.8 Daltons. The amino acid sequence for exendin(9-39) isshown as follows:H-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂.Exendin(9-39) comprises residues 9-39 of the GLP-1 receptor agonistexendin-4 and is a GLP-1 receptor antagonist. See, Montrose-Rafizadeh etal., Journal of Biological Chemistry, 272:21201-21206 (1997). As usedherein, the term “exendin(9-39)” encompasses pharmaceutically acceptablesalts of exendin(9-39), including but not limited to sulfate,hydrochloride, phosophate, sulfamate, acetate, citrate, lactate,tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, cyclohexylsulfamate and quinate salts. In someembodiments, exendin(9-39) is in the form of exendin(9-39) acetate orexendin(9-39) trifluoroacetate. Where not otherwise specified herein,exendin(9-39) acetate is used (obtained from Bachem (Clinalfa,Läufelfingen, Switzerland)).

In various embodiments, the GLP1A is a homologue, an analogue, or avariant of exendin(9-39). In various embodiments, the GLP1A is anyexendin(9-39) peptide known in the art. In various embodiments, theGLP1A is a derivative of an exendin-4 peptide other than exendin(9-39)or a derivative of a small molecule allosteric ligand (e.g. anyderivative of “compound 2”[6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline, a GLP1receptor agonist] that only binds one of the two allosteric bindingsites on the GLP1 receptor, or a similar derivative of BETP[4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]).In various embodiments, the GLP1A is a derivative of an endogenous GLP-1peptide (e.g. a derivative of GLP-1(1-36)NH2, GLP-1(1-37),GLP-1(7-36)NH2, GLP-1(7-37), or of oxyntomodulin). Derivatives ofparticular interest for these embodiments are those that have structurechanges or substitutions in any of the foregoing GLP-1 peptides,particularly those with changes or substitutions at one or more ofGLU16, VAL19, and ARG 20.

In all of these embodiments, the GLP1A acts not as an agonist but as anantagonist of the GLP-1 receptor. Some of these GLP1A, likeexendin(9-39) may also competitively antagonize the GIP receptor,although such activity is not believed to be required for efficacy.While the invention is not to be limited in any manner by virtue of aputative mechanism of action, molecules reported to be antagonists as aresult of binding only one of the two ligand binding sites of GLP-1receptor or otherwise having altered ligand-binding ability or affinity(e.g. at sites 149 or 333 on the GLP-1 receptor) resulting in attenuatedor inhibited GLP-1 receptor activation are suitable for use in themethods of the invention. In various embodiments, the GLP1A is resistantto proteolytic cleavage or to degradation by dipeptidyl peptidase-IV(DPPIV) or other dipeptidyl peptidase known in the art.

The present invention provides novel exendin(9-39) conjugates and newformulations of exendin(9-39) conjugates, in which the exendin(9-39) hasbeen chemically modified. For example, the present invention providesacylated exendin(9-39), which can be prepared, for example and withoutlimitation, with palmitic, lauric, lithocholic acid or a stearic diacidto prevent DPPIV degradation. Alternatively, the present inventionprovides conjugates in which the peptide is conjugated to an Fc proteinto minimize detection by the immune system, resulting in a longer actingdrug, at least for some patients. As another example, the presentinvention provides PEGylated exendin(9-39), a conjugate with apolyethylene glycol (PEG) so as to maximize plasma circulation timewhile minimizing loss of activity.

The methods, formulations, and examples discussed above are equallyapplicable to any GLP-1RAs having similar biochemical structure toexendin(9-39), including but not limited to other exendin analogues,pegylated versions, acylated versions, and amino acid variants thereof.

3.2 Patient Population

In some embodiments, a patient to be treated according to the methodsdescribed herein is a patient having hyperinsulinemic hypoglycemia (HH).In certain embodiments, the patient having hyperinsulinemic hypoglycemiahas previously had bariatric surgery (e.g., Roux-en-Y Gastric Bypass)and/or a related metabolic procedure. In certain embodiments, thepatient has previously had bariatric surgery (e.g., Roux-en-Y GastricBypass) and/or a related metabolic procedure and is at risk fordeveloping hyperinsulinemic hypoglycemia.

Patients with hyperinsulinemic hypoglycemia may be identified by anysuitable method. In some embodiments, hyperinsulinemic hypoglycemia isdiagnosed by the presence of Whipple's triad, which has the followingcriteria: (1) the occurrence of hypoglycemic symptoms; (2) documentedlow plasma glucose level at the type of the symptoms; and (3) resolutionof the symptoms after plasma glucose is raised. In some embodiments,hyperinsulinemic hypoglycemia is defined by the occurrence of capillaryglucose ≤50 mg/dL at least once per month by patient report or medicalrecord. In some embodiments, hyperinsulinemic hypoglycemia is defined bya plasma glucose concentration of ≤55 mg/dL during an oral glucosetolerance test in association with inappropriately elevated plasmainsulin (≥3 uU/mL) or c-peptide (>0.3 mg/dL) when glucose was ≤55 mg/dL.In some embodiments, hyperinsulinemic hypoglycemia is defined by aplasma glucose concentration of ≤60 mg/dL during an oral glucosetolerance test or meal tolerance test in association withinappropriately elevated plasma insulin (≥3 uU/mL) or c-peptide (>0.3mg/dL) when glucose was ≤60 mg/dL.

“Hyperinsulinemic hypoglycemia,” as used herein, encompasses theconditions dumping syndrome, nesideoblastosis, noninsulinomapancreatogenous hypoglycemia syndrome (NIPHS), and/or post-prandialreactive hypoglycemia. Hyperinsulinemic hypoglycemia may result from agastric or bariatric procedure, such as a Roux-en-Y gastric bypass(RYGB), or may have a congenital, acquired, or induced origin.

Those skilled in the art will appreciate that hyperinsulinemichypoglycemia is occasionally referred to as dumping syndrome,nesideoblastosis, noninsulinoma pancreatogenous hypoglycemia syndrome(NPHS), and/or post-prandial reactive hypoglycemia, and so may bereferred to herein as “hyperinsulinemic hypoglycemia.” Those skilled inthe art will further appreciate that hyperinsulinemic hypoglycemia mayresult from a gastric or bariatric procedure, or may have a congenital,acquired, or induced origin, and the methods and compositions may beused for therapeutic benefit with patients from any of these groups.

In one embodiment, the patient treated has previously had a bariatricand/or related metabolic procedure (of any type, including but notlimited to Roux-en-Y Gastric Bypass, Vertical Sleeve Gastrectomy,placement of an endosleeve device, such as the EndoBarrierGastrointestinal Liner System, also called an “endoluminal liner,”duodenal mucosal resurfacing, also referred to as duodenal ablation,partial bypass of the duodenum, involving duodeno-ileal orduodeno-jejunal anastomosis, vagal nerve blockade, and/or pyloroplasty)and so may be referred to herein as “post bariatric surgery” even thoughoften bariatric surgery may be thought of exclusively as weight losssurgery. Such procedures may be intended for weight loss or metabolicbenefit (such as resolution of diabetes), or both, and typically involveany of the foregoing: partially or completely bypassing the duodenumand/or decreasing nutrient exposure to the duodenum, increasing therapidity of nutrient transit to the lower part of the intestines (oftenspecifically the ileum), and/or otherwise increasing ileal nutrientexposure. All such weight loss or metabolic procedures, referred toherein as “bariatric procedures” may enhance secretion of GLP-1 from thedistal small intestine, especially the ileum, leading to elevatedinsulin secretion, and in some patients hypoglycemia. The methods andformulations of the invention are applicable to any of these conditionsand conditions of similar biologic origin or cause. The patient may bereferred to as a “post bariatric surgery” patient. or “post-RYGB.”

In another embodiment, the patient has previously had a relatedmetabolic procedure. As but one example, in one embodiment, the patienttreated has previously had a non-bariatric surgical procedure involvingthe gastrointestinal system (including but not limited to esophagectomy,for example for treatment of esophageal cancer, Nissen Fundoplication,for example gastroesophageal reflux, or gastrectomy, for example fortreatment of gastric cancer) and so may be referred to herein as “postgastrointestinal surgery.”

In another embodiment, the patient treated is prediabetic and/or insulinresistant and may benefit from prevention of pancreatic hyperstimulationfrom oral carbohydrate ingestion leading to post-prandial hypoglycemia.In another embodiment, a treated patient has a congenital, acquired, orinduced form of hyperinsulinemic hypoglycemia.

In a preferred embodiment, however, the patient has had bariatricsurgery to aid in weight loss and/or metabolic control and has sufferedhypoglycemic excursions requiring urgent medical attention; suchpatients, as demonstrated conclusively in the examples below, canbenefit markedly from treatment with a subcutaneously administeredformulation of exendin(9-39) in accordance with the invention.

A typical adult patient with hyperinsulinemic hypoglycemia will presentwithin 10 years of bariatric and/or other gastrointestinal surgery withsymptoms of hypoglycemia (e.g. palpitations, tremor, weakness, sweating,confusion, fatigue, blurred vision,) within 5 hours of eating that areassociated with a plasma glucose of ≤60 mg/dL and immediate resolutionwith carbohydrate intake. Many patients experience neuroglycopenicsymptoms, such as altered mental status, loss of consciousness, orseizures. Hyperinsulinemia (>2 uU/mL or 13.9 pmol/L) may be documentedin the proper laboratory setting at the time of the hypoglycemic event.However, documentation of hyperinsulinemia is not always possible due tologistical challenges associated with this testing (which involvesinduced hypoglycemia) and concerns over patient safety.

With the increasing incidence of obesity in children and adolescents,and the consequent increasing use of bariatric surgery in the pediatricand adolescent population, hyperinsulinemic hypoglycemia is anticipatedin this cohort, and will likely present similarly to the typical adultpatient.

Treatment in the typical adult or pediatric patient refers to treatmentsuch that the postprandial plasma glucose nadir is maintained above aconcentration of approximately 55 mg/dl (3.0 mmol/liter) based upon theEndocrine Society's Clinical Guidlines (Journal of ClinicalEndocrinology & Metabolism, March 2009, 94(3): 709-728), and symptoms ofhypoglycemia are reduced. Ideally, normal plasma glucose concentrationsare maintained, with those skilled in the art recognizing that in humansa blood glucose level of 65 mg/dl or greater is preferred.

Physicans skilled in the art will recognize from this disclosure thatthe methods of the invention provide effective treatment, such that aphysician following the same prescribing information herein can expecttherapeutic benefit will be achieved in patients whom, for treatment ofvarying underlying conditions, have had surgical manipulation of thegastrointestinal anatomy, and resultant secondary hyperinsulinemichypoglycemia. Accordingly, to illustrate, the methods of the inventioncan be used to treat patients such as: 1) a patient whom, due togastroesophageal reflux, underwent a Nissen Fundoplication procedure,and subsequently developed secondary hyperinsulinemic hypoglycemia; 2) apatient whom, due to a malignant gastric tumor (e.g. adenocarcinoma,gastrointestinal stromal tumor (GIST), or lymphoma), required partitialor complete gastrectomy, with or without any of the foregoing gastricreconstructive procedures: Bilroth I, Bilroth II, RYGB, or Jejunalinterposition, developed secondary hyperinsulinemic hypoglycemia; and 3)a patient whom, due to a tumor involving the esophagus or the esophagealgastric junction (EGJ), underwent an esophagectomy, developed secondaryhyperinsulinemic hypoglycemia.

Those skilled in the art will further appreciate that patients withhypoglycemia due to acquired or congenital hyperinsulinism (“endogenoushyperinsulinemia” as used herein refers to any such condition not causedby bariatric surgery or GI surgery) should benefit from application ofthe methods of the invention. Hypoglycemia in these instances can besevere, even life-threatening. Acquired hyperinsulinism may result frominsulinomas, autoimmune syndromes, reactive hypoglycemia, adultnesidioblastosis, or gastric dumping syndrome (not due to bariatric orGI surgery). Congential hyperinsulinism may manifest in the newbornperiod, or many years later. Accordingly, the methods and formulationsof the invention include methods to treat such conditions. In the caseof hyperinsulinemia resulting from an insulinoma and congenitalhyperinsulinism, a sustained release formulation and/or an immediaterelease formulation that is administered constistently, such as via asubcutaneous pump, would be employed, with particular emphasis on theprevention of nocturnal hyperinsulinemia.

In similar fashion, hyperinsulinism may further be induced as amedicinal side-effect of, for example, a GLP-1 agonist, such asexenatide, liraglutide, lixisenatide, albiglutide, and dulaglutide.Accordingly, the methods and formulations of the invention includemethods to treat overdoses with such drugs.

In some cases, patients with hyperinsulinemic hypoglycemia may alsopresent with cumulative hyperinsulinemic hypoglycemia-associatedcognitive impairment. Accordingly, the methods and formulations of theinvention include methods to treat or prevent a worsening of cognitiveimpairment in such patients. Further, in pediatric and adult patientsalike, acute and chronic hypoglycemia may be associated with morbiditiesnot only such as cognitive impairment, but also depression, heartpalpitations/tachycardia, and potentially other conditions, all of whichmay be reduced or prevented by preventing hypoglycemia by administrationof a GLP1A, such as exendin(9-39), as described herein forpost-bariatric patients suffering from hyperinsulinemia/hypoglycemia. Insome diabetic patients, severe hypoglycemia has repeatedly beenassociated with increased total and cardiovascular mortality risk. Thus,prevention of severe hypoglycemia is an important clinical goal for bothhospitalized and non-hospitalized patients, and the present inventionprovides methods and formulations useful for both groups of patients.

3.3 Treatment Parameters

In some embodiments, compositions comprising a therapeutically effectivedose of the GLP1A, such as exendin(9-39) or a homologue, analogue, orvariant thereof, are administered to a patient in need thereof for thetreatment or prevention of hyperinsulinemic hypoglycemia.

3.3.1 Administration Route

In some embodiments, the GLP1A is administered to a patient in needthereof by any suitable route of administration, such as subcutaneously,parenterally, transmusocally, transdermally, intramuscularly,intravenously, intra-dermally, intra-peritonealy, orally, or nasally.

3.3.1.1 Subcutaneous Administration

In some embodiments, the GLP1A, such as exendin(9-39) or a homologue,analogue, or variant thereof, is subcutaneously administered to apatient in need thereof. Sites of injection, include, but not limitedto, injection in the thigh, abdomen, upper arm region, or upper buttockregion.

While the present invention provides a variety of methods and materialsfor treatment and prevention of hyperinsulinemic hypoglycemia, oneaspect of this invention relates to the pharmaceutical compositions andmethods involving subcutaneous delivery of exendin(9-39) in dosestherapeutically effective for this indication. In many embodiments,these formulations for subcutaneous administration are formulated asimmediate release preparations, and are conveniently packaged, forexample, in the form of the dual-chamber pen device provided by theinvention, for patients or their care providers to administertherapeutically effective amounts in doses ranging from 2-100 mg.

3.3.1.2 Oral Administration

The present invention also provides GLP1A (e.g., exendin(9-39))compositions suitable for oral administration. Exendin(9-39) can beformulated for oral delivery with a formulation consisting a proteaseinhibitor to prevent digestion and an absorption enhancer to facilitatepassive diffusion through the intestine wall. The formulation can befilled into a capsule coated with an enteric coating using pH sensitivepolymers such as Eudragit® to protect from the acidic pH in the stomach(see, www.oramed.com/technology/scientific-abstracts/). In someembodiments an oral formulation of exendin(9-39) of the instantinvention combines an absorption-enhancing excipient, such as Eligen® soas to inhibit acid and peptidase-mediated degradation, and improvepassive transport across the enterocyte lumen and into the intracellularspace.

3.3.1.3 Inhaled Delivery

The present invention also provides methods and compositions in whichGLP1A (e.g., exendin(9-39)) is administered by inhalation. Some patientsmay prefer inhalation over subcutaneous or other forms of delivery, andthe present invention provides suitable dry powder inhalationformulations, generally constituting a room temperature stable powdercontaining exendin(9-39), that can be supplied in capsules and deliveredby a device, for example and without limitation, as used for therecently approved inhaled insulin, Afrezza (see,www.healthline.com/diabetesmine/welcome-afrezza-inhaled-insulin-gets-real#5).

3.3.2 Administration Dose

As discussed herein, patients with hyperinsulinemic hypoglycemia may betreated with a GLP1A, such as exendin(9-39) or a homologue, analogue, orvariant thereof, at a therapeutically effective dose of 2-100 mg, e.g.,at a dose ranging from 2-100 mg, 10-75 mg, 20-50 mg, 20-40 mg, 2-10 mg,2.5-10 mg, or 2.5-7.5 mg, depending upon the needs and physicalattributes of the patient. Exemplary doses include 10 mg, 12.5 mg, 15mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 35 mg, 40 mg, or 50mg. In some embodiments, the GLP1A (e.g., exendin(9-39)) is administeredat a dose of about 30 mg or lower (e.g., about 10 mg to 30 mg, 10 mg to25 mg, 10 mg to 20 mg, 15 mg to 20 mg, 10 mg to 17.5 mg, and 10 mg to 15mg). It will be understood by a person of ordinary skill in the art thatthe doses described herein can be administered at varyingconcentrations, including but not limited to the injectateconcentrations described in Section 3.3.4.1 below.

In some embodiments, exendin(9-39) is formulated and administered in aninjectable pen device that may be pre-programmed to deliver a fixeddosage amount ranging from 2-100 mg, 10-75 mg, 20-50 mg, 20-40 mg, 2-10mg, 2.5-10 mg, or 2.5-7.5 mg, depending upon the needs and physicalattributes of the patient. In some embodiments, adult patients (60-100kg or more) will receive therapeutic benefit from a single dose in therange of 10-75 mg, with some achieving full therapeutic effect with20-40 mg. While some or most adult patients will benefit from 10-30 mg,dosing may be initiated at lower doses, for example at 4-7 mg, 1-10 mg,or 2.5-7.5 mg, and maintained if efficatious or alternatively increasedstepwise if an enhanced dose response is desired. For especially smallpatients, for example in the case of children, or infants, a dose as lowas 1 mg or less might be employed. In some instances, for example in thecase of altered subcutaneous absorption or metabolism, doses outside ofthis range may be warranted. The foregoing dose ranges also guide dosingof other GLP1A with similar bioactivity, and those of skill in the artappreciate that simple tests can be performed to provide relativebioavailabilities of any two GLP1A, such that the specific informationprovided here re exendin(9-39) can readily be translated into any otherGLP1A the practitioner of the invention elects to employ.

3.3.3 Administration Schedule

In some embodiments, each dose will be administered in a total volumeranging from 0.25-2 ml injectate, with most patients administering aninjection volume ranging from 0.5-1.5 ml, i.e., 0.7-1 ml. In manyembodiments, patients will self-administer this dose at least once a day(qD), and often twice a day (BID). Some patients will administer thisdose with each meal, it being understood that “with each meal” typicallyrefers to a set period (at least 60 minutes, for example) before a meal,e.g., the first or last meal of the day.

In various embodiments, the invention provides immediate release anddelayed release formulations of exendin(9-39) and methods for theirdelivery by subcutaneous administration to post-bariatric surgerypatients suffering from hypoglycemia. In one embodiment of theinvention, the patient self-administers (or is wearing a deviceprogrammed to administer) the exendin(9-39) in a dose ranging from 4-40mg, 1-10 mg, or 2.5-10 mg, although many patients will receivetherapeutic benefit by a dose ranging from 10-25 mg or other dose asdescribed herein. Dosing will typically be qD or BID, with qDadministration typically utilizing a delayed or extended releaseformulation of the invention. The first dose may be administered in theevening, such that it provides protection during breakfast the followingday, with subsequent doses following the next evening and each eveningthereafter for qD administration or the following morning and abouttwelve hours later for BID administration.

3.3.3.1 QD and BID Administration

QD and BID administration are well known in the medical arts. In someembodiments BID doses are administered (e.g., self-administered) atabout 12 hour intervals (e.g., 7 a.m. and 7 p.m.). However, shorter(e.g., 8 a.m. and 6 p.m.) or longer (e.g., 7 a.m. and 10 p.m.) intervalsbetween BID administrations are possible provided the administrationsare at least about 6 hours apart. Preferably the BID administrations areat least about 7 hours, 8 hours, 9 hours, 10 hours or 11 hours apart.Preferably the BID administrations are not more than about 15 hoursapart.

In some embodiments QD doses are administered at about 24 hour intervals(e.g., 7 p.m. on successive days). However, shorter (e.g., 7 p.m. and 6p.m. on successive days) or longer (e.g., 7 p.m. and 10 p.m. onsuccessive days) intervals between QD administrations are possibleprovided the administrations are at least about 18 hours apart.Preferably the QD administrations are at least about 20 hours, 21 hours,22 hours, or 23 hours apart. Preferably the QD administrations are notmore than about 30 hours apart.

3.3.3.2 Timing of Adminstration and Relationship to Meals

In one aspect of the invention, an immediate-release formulation of theGLP1A (e.g., exendin(9-39)) is provided as a subcutaneous injectableformulation that is administered prior to the administration of a meal.For example, in some embodiments, the GLP1A is administered within60-150 minutes (e.g., within 90-120 minutes) prior to morning andevening meals (or before the two main meals of the day, approximately 6hours or more apart). In one embodiment, the GLP1A is administered BIDas morning and evening administration, with patients dosing 60-150minutes (e.g., 90-120 minutes) prior to the morning and evening meals,so that the peak GLP-1 plasma concentration occurring approximately30-60 minutes post-meal will be countered by sufficient GLP1A(exendin(9-39)) plasma concentrations at that time to prevent GLP-1induced hyperinsulinemia.

In another embodiment the BID dosing will be a morning and eveningadministration with a morning administration after wakening in themorning and evening administration about 12 hours later (in someembodiments, about 12-14 hours or about 12-16 hours later). The morningadministration may be before or after breakfast. In this embodiment, thedosing schedule is independent of (i.e., not based on, or dictated by)the timing of meals. In some embodiments the morning administration iswithin a specified time before and/or after the morning meal (e.g. onehour before and/or one hour after breakfast). In some embodiments themorning administration is before or after the morning meal, as discussedabove, and the evening administration is prior to retiring for the night(bedtime) such as between the evening meal and bedtime, or within 1, 2,or 3 hours of bedtime.

In a related embodiment the dosing schedule is semi-independent ofmealtimes. For example, in the semi-independent schedule the morningdose is administered on a predetermined schedule relative to the morningmeal and the evening dose is scheduled at a time independent of the timeof the evening meal (e.g., about 12 hours after the morningadministration without regard to the time of the evening meal).

Without intending to be bound by any specific mechanism, it is believedthat the schedule, dose, route and formulations of the invention allowthe evening administration to provide additional protection atbreakfast, and the morning administration to provide protection duringthe day (e.g., lunch, dinner, or multiple small meals during the day).Advantageously, subcutaneous BID administration of a therapeuticallyeffective dose of GLP1A (e.g., exendin(9-39)) is protective even whennot timed to coincide with meals. In contrast, as demonstrated inExample 2, an IV bolus injection of 7,500 pmol/kg exendin(9-39) reversedhypoglycemia only if timed to coincide with the expected peak GLP-1plasma concentrations.

In some embodiments, the GLP1A is administered QD as an eveningadministration, with the administration scheduled at a time independentof the time of meals (e.g., independent of the timing of the eveningmeal). In some embodiments, the evening administration is prior toretiring for the night (bedtime) such as between the evening meal andretiring, or within 1, 2, or 3 hours of retiring. In another embodimentthe GLP1A is administered QD as a morning administration, with patientsdosing 60-150 minutes (e.g., 90-120 minutes) prior to the morning meal,so that the peak GLP-1 plasma concentration occurring approximately30-60 minutes post-meal will be countered by sufficient GLP1A(exendin(9-39)) plasma concentrations at that time to prevent GLP-1induced hyperinsulinemia.

3.3.4 Administration Formulation

3.3.4.1 Injectate Concentration and Volume

Surprisingly, certain pharmacokinetic parameters of subcutaneous GLP1A(e.g., exendin(9-39)) administration can be modified by selecting theconcentration of the GLP1A in the injectate. As described in theExamples, subcutaneous injection of a low concentration formulationresults in a shorter Tmax (i.e., a faster rise to Cmax) relative to ahigher concentration. Subcutaneous injection of a high concentrationformulation results in a lower Cmax, a longer Tmax, and longer half-liferelative to a lower concentration. See FIG. 7A and Table 4.

For the purposes of this invention a concentration less than 20 mg/ml isa “low” concentration, e.g., 4-20 mg/ml, preferably about 10-20 mg/ml,and often about 8-16 mg/ml, most often about 13-16 mg/ml, and very often15 mg/ml. The low concentration formulation results in a pharmacokineticprofile useful for BID administration. As shown in FIG. 7B and Table 5(Example 3), subcutaneous administration of exendin(9-39) at varyingdoses but equivalent concentrations of about 13-16 mg/ml resulted in afavorable immediate release formulation with a Cmax greater than apreferred steady state plasma exendin(9-39) concentration of at least100 ng/ml as measured by liquid chromatograph-mass spectrometry, at 10,20 and 30 mg doses.

For purposes of this invention a concentration greater than about 20mg/ml (e.g., 20-45 mg/ml) is considered a “high” concentration. As shownin FIG. 7A and Table 4, subcutaneous injection of a relatively moreconcentrated solution, for example in a range inclusive of and exceeding20 mg/ml, e.g., 20-40 mg/ml, will result in a lower Cmax, with a longerhalf-life. For example, as shown in FIG. 7A and Table 4, subcutaneousadministration of exendin(9-39) at a dose of about 20 mg andconcentration of about 24 mg/ml exhibited a significantly longerhalf-life than subcutaneously administered exendin(9-39) at a dose ofabout 10 mg and concentration of about 16 mg/ml (9.14 hours vs. 3.60hours). In some embodiments, a more highly concentrated solution ofexendin(9-39) results in an exendin(9-39) plasma Cmax that is lower thana relatively lower concentration formulation but which is still greaterthan a preferred steady state plasma exendin(9-39) concentration of 70ng/ml or greater (e.g., as shown in FIG. 7A and Table 4 for the “10×”(approximately 20 mg) dose as compared to the “5×” (approximately 10 mg)dose). Thus, a more concentrated solution may be more amenable to lessfrequent dosing, e.g., QD dosing, or to BID dosing that is not tied tomeals.

The examples below demonstrate that subcutaneous injection of arelatively dilute solution, for example of 4-20 mg/ml, and most often8-16 mg/ml, will result in a pharmacokinetic profile amenable to BIDadministration (see FIG. 7B and Table 5). In one embodiment the BIDdosing will be a morning and evening administration with patients dosingupon awakening in the morning and 12 hours later, i.e. dosing will notbe meal-based. In one embodiment, the BID dosing will involve morningand evening administration, with patients dosing 60-150 minutes prior tothe morning and evening meals, so that the peak GLP-1 plasmaconcentration occurring approximately 30-60 post-meal will be counteredby sufficient GLP1A plasma concentrations at that time to prevent GLP-1induced hyperinsulinemia.

The examples below also demonstrate that subcutaneous injection of amore concentrated solution, for example in the range inclusive of andexceeding 20 mg/ml will result in a more sustained releasepharmacokinetic profile, with a longer half-life, and thus may be moreamenable to less frequent dosing, e.g., qD dosing or BID dosing that isnot tied to timing of meals. See, FIG. 7A and Table 4.

In some embodiments, the GLP1A such as exendin(9-39) is administered(e.g., subcutaneously administered) at a concentration of about 4-25mg/ml, about 4-20 mg/ml, about 10-25 mg/ml, about 10-20 mg/ml, about10-18 mg/ml, about 8-16 mg/ml, about 12-20 mg/ml, about 10-15 mg/ml, orabout 13-16 mg/ml (e.g., about 4 mg/ml, about 5 mg/ml, about 6 mg/ml,about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml,about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml,or about 25 mg/ml).

In some embodiments, the GLP1A is administered (e.g., subcutaneouslyadministered) at a concentration in the range of about 13 mg/ml to about16 mg/ml. In some embodiments, the GLP1A is administered at aconcentration of about 15 mg/ml.

As shown in FIG. 7B and Table 5, both a relatively lower dose of 10 mgand a relatively higher dose of 30 mg yielded a Cmax greater than thepreferred steady state plasma exendin(9-39) concentration of 70 ng/ml orgreater and were efficacious in reversing hyperinsulinemic hypoglycemiawhen administered at approximately equal concentrations in the range ofabout 13-16 mg/ml. Thus, in one aspect, a relatively lower dose of aGLP1A such as exendin(9-39) (e.g., a dose of about 5-10 mg, e.g., about5 mg, about 7.5 mg, or about 10 mg) can be efficacious in treatinghyperinsulinemic hypoglycemia by adjusting the GLP1A solution to anappropriate concentration as described herein. In some embodiments, arelatively lower dose of a GLP1A such as exendin(9-39) (e.g., a dose ofabout 5-10 mg, e.g., about 5 mg, about 7.5 mg, or about 10 mg) isadministered at a concentration of at least about 10 mg/ml, e.g., at aconcentration in the range of about 13-16 mg/ml, e.g., at aconcentration of about 15 mg/ml.

Generally a GLP1A such as exendin(9-39) is administered at aconcentration sufficient to result in a steady state plasmaconcentration (e.g., exendin(9-39) concentration) of at least 70 ng/ml,at least 100 ng/ml, or at least 150 ng/ml as measured by liquidchromatograph-mass spectrometry. In some embodiments, the GLP1A (e.g.,exendin(9-39)) is administered at a concentration sufficient to resultin a steady state plasma exendin(9-39) concentration of about 100-200ng/ml. In some embodiments, the GLP1A (e.g., exendin(9-39)) isadministered at a concentration sufficient to result in a steady stateplasma exendin(9-39) concentration of at least 70 ng/ml up to 250 ng/ml.

Table A below provides exemplary therapeutically effective GLP1 (e.g.,exendin(9-39)) formulations:

TABLE A GLP1A Concentration 1 10-30 mg 10-25 mg/mL 2 10-30 mg 10-20mg/mL 3 10-30 mg 10-18 mg/mL 4 10-30 mg 13-16 mg/mL 5 10-25 mg 10-25mg/mL 6 10-25 mg 10-20 mg/mL 7 10-25 mg 10-18 mg/mL 8 10-25 mg 13-16mg/mL 9 10-20 mg 10-25 mg/mL 10 10-20 mg 10-20 mg/mL 11 10-20 mg 10-18mg/mL 12 10-20 mg 13-16 mg/mL 13 10-15 mg 10-25 mg/mL 14 10-15 mg 10-20mg/mL 15 10-15 mg 10-18 mg/mL 16 10-15 mg 13-16 mg/mL 17 15-30 mg 10-25mg/mL 18 15-30 mg 10-20 mg/mL 19 15-30 mg 10-18 mg/mL 20 15-30 mg 13-16mg/mL 21 15-25 mg 10-25 mg/mL 22 15-25 mg 10-20 mg/mL 23 15-25 mg 10-18mg/mL 24 15-25 mg 13-16 mg/mL 25 15-20 mg 10-25 mg/mL 26 15-20 mg 10-20mg/mL 27 15-20 mg 10-18 mg/mL 28 15-20 mg 13-16 mg/mL

In some embodiments, each administration of the GLP1A (e.g., each BIDsubcutaneous administration of exendin(9-39)) results in a GLP1A Cmax(e.g., exendin(9-39) Cmax) of at least 100 ng/ml. In some embodiments,for example for patients having relatively higher GLP-1 levels or havinggreater beta-cell sensitivity to GLP-1, each administration of the GLP1A(e.g., each BID subcutaneous administration of exendin(9-39)) results ina Cmax of at least 150 ng/ml.

In some embodiments, the GLP1A injectate comprises a GLP1A (e.g.,exendin (9-39)) dose and concentration that, when administered, resultsin steady state plasma GLP1A concentration of at least 70 ng/ml,preferably at least 100 ng/ml, or even more preferably at least 150ng/ml, as measured by LCMS. In some embodiments, the GLP1A (e.g.,exendin(9-39)) formulation has such a dose and concentration thatresults in steady state plasma concentration of 100-250 ng/ml, e.g.,100-200 ng/ml, 100-150 ng/ml, or 150-200 ng/ml.

In some embodiments, each dose is administered in a total volume rangingfrom 0.25-2 ml injectate, with most patients administering an injectionvolume ranging from 0.5-1.5 ml, e.g., 0.7-1 ml.

3.3.4.2 Injectate Formulation

The GLP1A may be administered in any pharmaceutically acceptable form.In some embodiments, a GLP1A such as exendin(9-39) is formulated with apharmaceutically acceptable diluent or carrier that is suitable forsubcutaneous administration. Examples of pharmaceutically acceptablediluents or carriers include, but are not limited to, water, saline, andisotonic buffer solutions. In some embodiments the injectate formulationfurther comprises one or more additional excipients such aspreservatives and pH adjustment agents.

In one approach the GLP1A such as exendin(9-39) is formulated in normalsaline (0.9% saline). In one approach the GLP1A is formulated with anantimicrobial preservative, a tonicity-adjusting agent, such asmannitol, and/or a buffer (e.g., to bring the solution to a pH of about4-5).

As shown in Example 3 and FIG. 7A, administration of a lower dose (10mg, or “5×”) resulted in a higher exendin(9-39) Cmax than higher dosesformulated in the same volume of solution (i.e., having a higherconcentration). Without intending to be bound by a particular mechanism,this may be a result of aggregation (e.g., dimer or higher multimerformation). Thus, in one approach the GLP1A is formulated with an agentto reduce aggregation or dimer formation such as a surfactant (e.g., anon-ionic surfactant, such as a polysorbate or a poloxamer), polyol, orsugar, or by optimizing the pH and/or ionic strength of the solution.

The present invention provides a variety of different formulations,including but not limited to formulations of exendin(9-39) and otherGLP-1 antagonists of similar structure and biological activity thatprovide for more extended release.

3.3.4.2.1 Immediate Release (IR) Formulations

In one aspect, the GLP1A (e.g., exendin(9-39)) is formulated forimmediate release. In one aspect of the invention, an immediate-releaseformulation of exendin(9-39) is provided as a subcutaneous injectableformulation that is administered within 60-150 minutes prior to morningand evening meals (or before the two main meals of the day,approximately 6 hours or more apart). In one aspect of the invention,the immediate-release formulation of exendin(9-39) is administered as asubcutaneous injection in the abdomen, thigh, or upper arm.

In one embodiment, the present invention provides injectable,immediate-release formulations of exendin(9-39), and variousderivatives, homologues, and analogues of exendin(9-39), as discussedabove, and other GLP-1RAs of similar activity and/or structure toexendin(9-39), wherein said injectable formulations are equivalent toformulations used to deliver exenatide, marketed as BYETTA™ (see U.S.Pat. Nos. 5,424,286; 6,858,576; 6,872,700; 6,902,744; 6,956,026;7,297,761; 7,521,423; 7,741,269, incorporated herein by reference).

Lyophilized peptide in dual-chamber pen device or vial/syringe device:While exendin(9-39) can be formulated in any way suitable foradministration, a suitable immediate release pharmaceutical compositionfor subcutaneous injection in accordance with the invention containsfrom about 2.5-40 mg/mL of exendin(9-39) in normal saline or otherpharmacologically acceptable liquid or suspension formulation, and maybe administered in one or more injections. Such composition will beespecially convenient for administration of doses that total about 0.01to 1 mg/kg body weight, administered by subcutaneous injection in theabdomen, thigh, or upper arm within 1 minute to about 2 hours of meals,with dosing frequency limited to up to 3 times daily, i.e., no more thanTID administration. Most patients, however, will benefit from BIDadministration, which as shown in the examples below can be efficaciouswhen administered as a relatively simply immediate release formulation,and as in the results shown for a more concentrated formulation withlonger-acting pharmacokinetics, qD administration will be beneficial formany patients, particulary when an extended or sustained releaseformulation is administered. While some patients will be administered astandard dose (e.g., 2.5, 5, 10, 20, or 30 mg of exendin(9-39), forexample), in other patients, dosing will be initiated at a low dose, andincreased stepwise if a lower dose does not result in acceptableglycemic control. The drug product can be supplied as a freeze-driedlyophilized powder, stored in a 1 to 3 mL or larger, e.g., 5 mL,dual-chamber cartridge that is compatible with a disposable pen injector(see, for example, the Ypsomed dual chamber cartridge/pen injector:http://www.ypsomed.com/yds/products/dual-chamber-pens.html).Two-to-three dose strengths can be conveniently made available topatients, including for example doses in the range of 2-100 mg ofexendin(9-39), to be reconstituted in a volume of 0.25-2.0 ml normalsaline per dose, or other pharmaceutically acceptable diluent suitablefor subcutaneous administration. Typical doses (dose strengths) include,for example and without limitation, 2.5 mg, 5 mg, 10 mg, 20 mg, and 30mg of exendin(9-39) for immediate and extended release formulations. Toachieve a more concentrated solution, for example for a more slowrelease effect, the same doses may be reconstitution in a smallervolume, for instance in as low as 0.15-0.25 ml. Overall, theconcentration for the final reconstituted drug product may range from2-40 mg/ml, but more often from 5-30 mg/ml. The present inventionprovides a portable device that permits patients to reconstitute, prime,and inject the lyophilized peptide conveniently “on the go,” for examplein association with meals. The present invention also provides suchcompositions in a unit, a single-dose, or a multi-dose glass vial orampule for administration with the use of a pre-filled syringecontaining normal saline or other pharmacologically acceptable liquid orsuspension formulation, for use in solubilizing/diluting andsubsequently administering a single injection subcutaneously using aninsulin syringe. Depending on the dose and concentration of the desiredfinal reconstituted drug product, the number of doses provided couldrange from 1-40 doses. For example, a 5 mg dose that employs aconcentration of 15 mg/ml (equating to 5 mg in 0.33 ml) would provide upto 15 doses, whereas a 10 mg dose at the same concentration would yielda total of 7 doses. If higher concentrations are employed, the number ofpotential doses provided may increase. In all instances, the use of anantimicrobial or other preservative may be employed, to maintainsterility of the reconstituted drug product if intended for more thansingle day use.

Sterile (preserved) solution in pen-injector device or in glass vial orampule and syringe (kit): The present invention also providesexendin(9-39) drug product formulated for subcutaneous injection as asterile, preserved isotonic solution in a glass cartridge pen-injectordevice. Such compositions for example and without limitation contain2-80 mg of exendin(9-39), an appropriate volume of an antimicrobialpreservative, a tonicity-adjusting agent, such as mannitol, and a bufferto bring the solution to a pH of about 4-5. As noted, typical dosesinclude 2.5 mg, 5 mg, 10 mg, 20 mg, and 30 mg. A prefilled pen can bemade in accordance with the invention to deliver a unit dose of anydesired amount, e.g., 5 to 40 mg, e.g., 10 to 30 mg, being typicalamounts that could be administered to an adult human. The presentinvention also provides such compositions in a unit or multi-dose glassvial or ampule for administration with the use of a syringe, similar tothe glucagon emergency kit.

3.3.4.2.2 Extended Release (ER) Formulations

In one aspect, the present invention relates to a GLP1A, such asexendin(9-39), formulated for extended release, i.e., an extendedrelease formulation, such that, when administered, the formulationensures that the active drug product has a lasting presence in the bloodthroughout the targeted time period in the course of treatment. Use ofthese formulations and methods allows plasma glucose homeostasis to bemaintained with fewer subcutaneous injections, relative to immediaterelease formulations. Various embodiments of this aspect of theinvention are described below.

Encapsulation with Microspheres and Nano-Lipocapsules: A GLP1A (e.g.,exendin(9-39)) can be formulated in accordance with the invention asslowly eroding microspheres. Such microspheres include, for example andwithout limitation, those made with a biopolymer, such as Poly(lactic-co-glycolic acid) (PLGA) or its equivalent. Such formulationsprovide for release of drug over an extended period of time (1-10weeks). To prepare the formulation, exendin(9-39) is loaded into themicrospheres, and the formulation provides that exendin is steadilyreleased over time as the matrix materials degrade. These microspherescan be formulated to minimize drug bursts and maintain a steady releaseprofile. Alternatively, exendin(9-39) is encapsulated intonano-lipocapsules to prepare another formulation of the invention, whichprovides similar sustained and extended drug release. These formulationsare provided in a variety of particle and capsule sizes andcompositions, providing the physician a variety of rapid, medium, andslow release profile formulations to optimize therapy for individualpatients.

In one embodiment, the present invention provides injectable,long-acting formulations of exendin(9-39), and various derivatives,homologues, and analogues of exendin(9-39), as discussed above, andother GLP-1RAs of similar activity and/or structure to exendin(9-39),wherein said long-acting formulations are equivalent to formulationsused to deliver exenatide, marketed as BYDUREON™ (see U.S. Pat. Nos.5,424,286; 6,479,065; 6,495,164; 6,667,061; 6,824,822; 6,858,576;6,872,700; 6,956,026; 7,223,440; 7,456,254; 7,563,871; 7,612,176;7,741,269; 8,216,180; 8,329,648; 8,431,685; 8,439,864; 8,461,105; and8,906,851, incorporated herein by reference), or equivalent toformulations used to deliver liraglutide, delivered in a daily dose,marketed as Victoza™ (see U.S. Pat. Nos. 6,004,297; 6,268,343;6,458,924; 7,235,627; 8,114,833; and 8,846,612).

In one aspect of the invention, the extended-release formulation ofexendin(9-39) is provided as a subcutaneous injectable formulation thatis administered once daily. In one aspect of the invention, anextended-release formulation of exendin(9-39) is provided as asubcutaneous injectable formulation that is administered once everyseven days, at any time of day, and with or without meals. In one aspectof the invention, the extended-release formulation of exendin(9-39) isadministered as a subcutaneous injection in the abdomen, thigh, or upperarm region. In one aspect of the invention, the extended-releaseformulation of exendin(9-39) is administered immediately after the doseis prepared. In some instances, the extended-release formulation ofexendin(9-39) is provided as an injectable suspension in a single-dosetray containing a vial of exendin(9-39), a vial connector, a prefilleddiluent syringe, and one or more needles. In some instances, theextended-release formulation of exendin(9-39) is provided as aninjectable suspension in a single-dose pen containing exendin(9-39), adiluent, and one or more needles.

Other formulations of the invention having a variety of differentfeatures and advantages may be made in accordance with the teachings ofU.S. Pat. Nos. 8,445,647; 8,895,033; 8,969,293; 8,299,025; 8,546,326;2015/0258016; 2015/0238568; 2015/0057227; 2015/0056285; 2014/0309168;2014/0256626; 2013/0252894; 2013/0195939; 2013/0172250, incorporatedherein by reference, substituting exendin(9-39) or other biochemicallysimilar GLP-1RA as described herein for the active pharmaceuticalingredient described.

In some embodiments, the GLP1A is formulated as a sterile, preservedisotonic solution in a unit or multi-dose glass vial or ampule foradministration with the use of a syringe, similar to the glucagonemergency kit. In some embodiments, the GLP1A is provided as aninjectable suspension in a single-dose tray containing a vial of GLP1A,a vial connector, a prefilled diluent syringe, and one or more needles.

In some embodiments, the GLP1A is formulated as a sterile, preservedisotonic solution in a glass cartridge pen-injector device. Suchcompositions, for example and without limitation contain 5-20 mg ofGLP1A, an appropriate volume of an antimicrobial preservative, atonicity-adjusting agent, such as mannitol, and a buffer to bring thesolution to a pH of about 4-5.

In some instances, the formulation of GLP1A is provided as an injectablesuspension in a single-dose pen containing GLP1A, a diluent, and one ormore needles.

3.3.5 Different Evening and Morning Injectates

In some embodiments, twice-per-day administration comprisesadministering a morning injectate and an evening injectate that containdifferent GLP1A (e.g., exendin(9-39)) doses and/or differentconcentrations of the GLP1A (e.g., exendin(9-39)). Generally, each ofthe injectates has a GLP1A amount and concentration within the rangesdescribed herein. However, in this embodiment, the amount of GLP1A inthe evening administration is greater than the amount in the morninginjectate and/or the GLP1A concentration in the evening injectate isgreater than the concentration of GLP1A in the morning injectate. Insome embodiments the two injectates will have different quantities, thesame concentration, of. In some embodiments the two injectates will havethe same amount of GLP1A but different concentration. In someembodiments both the concentration and amount of GLP1A will bedifferent.

Without intending to be bound by a particular mechanism, the increasedamount of GLP1A administered in the evening may provide higher GLP1Alevels at the time of the morning meal. Without intending to be bound bya particular mechanism, the increased concentration of GLP1A is expectedto result in a more “flat” plasma concentration profile, including alonger time to Tmax, for a more sustained effect at the time of themorning meal.

In some embodiments the amount of GLP1A (e.g., exendin(9-39)) in theevening injectate is 5 mg to 10 mg greater than the amount in themorning injectate. In some embodiments the amount of GLP1A in theevening injectate is 5 mg greater than the amount in the morninginjectate. In some embodiments the amount of GLP1A in the eveninginjectate is 10 mg greater than the amount in the morning injectate. Insome embodiments the amount of GLP1A in the morning injectate is 10 mg,15 mg, or 20 mg.

In some embodiments the concentration of GLP1A (e.g., exendin(9-39)) inthe evening injectate is 5 mg/ml-10 mg/ml greater than the amount in themorning injectate. In some embodiments the concentration of GLP1A in theevening injectate is about 5 mg/ml greater than the amount in themorning injectate. In some embodiments the concentration of GLP1A in theevening injectate is about 10 mg/ml greater than the amount in themorning injectate. In some embodiments the concentration of GLP1A in themorning injectate is 10-16 mg/ml and the concentration of GLP1A in theevening injectate is higher and is in the range of 15-20 mg. In somecases, the amount of GLP1A in the evening injectate is 5 mg-10 mggreater than the amount in the morning injectate. In some such cases theamount of GLP1A in the morning injectate is 10 mg and the amount in theevening injectate is 15 mg. In some such cases the amount of GLP1A inthe morning injectate is 10 mg and the amount in the evening injectateis 20 mg. In some such cases the amount of GLP1A in the morninginjectate is 15 mg and the amount in the evening injectate is 20 mg. Insome such cases the amount of GLP1A in the morning injectate is 15 mgand the amount in the evening injectate is 25 mg. In some such cases theamount of GLP1A in the morning injectate is 20 mg and the amount in theevening injectate is 25 mg. In some such cases the amount of GLP1A inthe morning injectate is 20 mg and the amount in the evening injectateis 30 mg.

In some cases, the concentration of GLP1A (e.g., exendin(9-39)) in themorning injectate is not the same as the concentration of GLP1A in theevening injectate. For example, in one approach the concentration ofGLP1A in the morning injectate is 15 mg/ml and the concentration ofGLP1A in the evening injectate is 20 mg/ml.

In some cases the GLP1A amount and concentration of the morning andevening injectates are selected such that the GLP1A Tmax after theevening administration is longer than the Tmax after the morningadministration.

In one approach, the evening injectate is prepared or formulated tofavor multimerization (e.g., dimerization) or precipitation of theGLP1A. Administration of the injectate at bedtime can delay absorption,producing a slower release profile compared to the morningadministration, resulting in an advantageous basal morning level of atleast 30 ng/mL Methods for preparing compositions comprisingmultimerized proteins are known. For example, the addition of a basicprotein, such as protamine, to the GLP1A preparation can favor formationof multimer peptide configurations. Alternatively, multimerization canbe achieved by precipitating the GLP1A out of solution, for examplethrough the addition of salts, such as zinc salts, such that the molarratio of the salt with respect to GLP1A is greater than 1, so as toreduce the solubility of the GLP1A in a neutral solvent. In thisapproach raising the pH (for example to 7.4), in the presence of suchsalts, can be used to favor precipitation of the peptide. Thus, in someembodiments the level of aggregation or multimerization in the eveninginjectate is greater than the level in the morning injectate. In someembodiments the GLP1A is in a less soluble form in the evening injectatecompared to the morning injectate.

In some embodiments an immediate release formulation (e.g., as describedherein) is administered in the morning and an extended releaseformulation (e.g., as described herein) is administered in the evening(e.g., before bedtime).

3.3.6 Duration of Therapy

Patients may receive therapy for a predetermined time, an indefinitetime, or until an endpoint is reached. Treatment may be continued on acontinuous daily or weekly basis for at least two to three months, sixmonths, one year, or longer. In some embodiments, therapy is for atleast 30 days, at least 60 days, at least 90 days, at least 120 days, atleast 150 days, or at least 180 days. In some embodiments, treatment iscontinued for at least 6 months, at least 7 months, at least 8 months,at least 9 months, at least 10 months, at least 11 months, or at leastone year. In some embodiments, treatment is continued for the rest ofthe patient's life or until administration is no longer effective inmaintaining normal plasma glucose levels to provide meaningfultherapeutic benefit. In some embodiments, adult patients (60-100 kg ormore) will receive therapeutic benefit from a single dose of GLP1A.

3.4 Delivery Devices

Devices such as injectable pen devices and pumps suitable forsubcutaneous injections are well known. Such devices may be used todeliver the GLP1A (e.g., exendin(9-39)) formulations describedhereinabove according to the methods described herein.

3.4.1 Subcutaneous Pump

In one embodiment, the method of the invention is practices with use ofa subcutaneous pump, and the invention provides such pumps containingthe GLP1A, e.g., exendin(9-39) or a conjugate thereof, formulated asdescribed herein for subcutaneous delivery. This methodology can confertighter plasma glucose control for some patients and is generally veryconvenient for the patient. Compositions for such method provided by theinvention include solution formulations and freeze dried lyophilizedpowder for reconstitution. See, e.g., Kumareswaran et al., DiscoveryMedicine, 2012, 13:159-170, incorporated by reference herein.

3.4.2 Injectable Pen Device

In some embodiments, GLP1A (e.g., exendin(9-39)) is administered usingan injectable pen device that may be pre-programmed to deliver a fixeddosage amount. In some embodiments, the device is pre-programmed todeliver a fixed dosage ranging from 5-30 mg, e.g., 10-20 mg, or 7.5-15mg, depending upon the needs and physical attributes of the patient. Insome embodiments, the GLP1A (e.g., exendin(9-39)) is formulated as animmediate release preparation, and is packaged, for example, in the formof a single or dual-chamber pen device (e.g., a 1 to 5 mL dual chamberpen—either a disposable pen or one that reloads disposable cartridges).

The drug product can be supplied as a freeze-dried lyophilized powder,stored in a 1 to 3 mL or larger, e.g., 5 mL, dual-chamber cartridge thatis compatible with a disposable pen injector (see, for example, theYpsomed dual chamber cartridge/pen injector:www.ypsomed.com/yds/products/dual-chamber-pens.html). Two-to-three dosestrengths can be conveniently made available to patients, including forexample doses in the range of 5-30 mg of exendin(9-39), to bereconstituted in a volume of 0.25-2.0 ml normal saline per dose, orother pharmaceutically acceptable diluent suitable for subcutaneousadministration.

In some embodiments, the drug product is supplied as individualinjectable pen devices that are pre-programmed to deliver a fixed dosageamount, in which the morning dosage amount and the evening dosage amountare different amounts and/or concentrations. For example, in someembodiments, a first pen (e.g., for morning administration) delivers adose in the range of 5-15 mg (e.g., a dose of 5 mg, 7.5 mg, 10 mg, 12.5,or 15 mg) and a second pen (e.g., for evening administration) delivers ahigher dose in the range of 15-20 mg (e.g., a dose of 15 mg, 17.5 mg, or20 mg). In some embodiments, the first pen delivers a dose of 10 mg andthe second pen delivers a dose of 15 mg. In some embodiments a first pendelivers a dose of 15 mg and the second pen delivers a dose of 20 mg.

In another aspect, the present invention provides kits comprisingindividual injectable pen devices as described herein. In someembodiments, a kit comprises a plurality of individual injectable pendevices (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pens in a kit). Insome embodiments, the kit comprises two or more individual injectablepen devices that are pre-programmed to deliver a fixed dosage amount, inwhich the morning dosage amount and the evening dosage amount aredifferent amounts and/or concentrations. For example, in someembodiments, the kit comprises a first pen (e.g., for morningadministration) that delivers a dose in the range of 5-15 mg (e.g., adose of 5 mg, 7.5 mg, 10 mg, 12.5, or 15 mg) and further comprises asecond pen (e.g., for evening administration) that delivers a higherdose in the range of 15-20 mg (e.g., a dose of 15 mg, 17.5 mg, or 20mg). In some embodiments, the kit comprises a first pen that delivers adose of 10 mg and a second pen that delivers a dose of 15 mg. In someembodiments, the kit comprises a first pen that delivers a dose of 15 mgand a second pen that delivers a dose of 20 mg.

3.5 Treatment Outcomes

In some embodiments, patients treated with the compositions and methodsdescribed herein exhibit an improvement in one or more symptoms ofhypoglycemia, including but not limited to neuroglycopenic symptoms,beta-adrenergic symptoms, or plasma glucose levels.

In some embodiments, treatment in the typical adult or pediatric patientrefers to treatment such that the postprandial plasma glucose nadir ismaintained above a concentration of approximately 55 mg/dl (3.0mmol/liter) based upon the Endocrine Society's Clinical Guidelines(Journal of Clinical Endocrinology & Metabolism, March 2009, 94(3):709-728), and symptoms of hypoglycemia are reduced. Ideally, normalplasma glucose concentrations are maintained, with those skilled in theart recognizing that in humans a blood glucose level of 65 mg/dl orgreater is preferred.

In some embodiments, treatment in a patient refers to treatment suchthat at least a 15% increase in postprandial plasma glucose nadir isachieved relative to baseline (e.g., before the onset of treatment). Insome embodiments, treatment in a patient refers to treatment such thatfor a patient having a postprandial plasma glucose nadir ≤50 mg/dl atbaseline (e.g., before the onset of treatment), an increase inpostprandial plasma glucose nadir to ≥55 mg/dl is achieved relative tobaseline.

Plasma glucose nadir can be measured, for example, by oral glucosetolerance test (OGTT) or meal tolerance test (MTT) as described herein.In some embodiments, treatment in a patient refers to treatment suchthat a statistically significant decrease in the severity of one or moresymptoms of hypoglycemia overall during a OGTT or MTT and/or ofneuroglycopenic symptoms elicited during the glucose “fall” period ofOGTT or MTT is achieved relative to baseline (e.g., before the onset oftreatment).

3.6 Co-Administration with an Amylinomimetic or Other Gastric EmptyingAgent

Some patients may enjoy added therapeutic benefit from theco-administration, in accordance with the invention, of anamylinomimetic or other agent that delays gastric emptying withexendin(9-39) or other GLP1A, as described herein. Delayed nutrienttransit due to co-administration of such an agent can diminish thepostprandial glucose peak, therein reducing the peak-to-nadir plasmaglucose drop, and the rate at which this drop occurs. While not to belimited or construed by any theory, one insight that lead to theinvention was the discovery that the plasma glucose concentration itselfdid not appear to be the primary elicitor of the early symptoms ofhypoglycemia; instead, the rate at which the glucose decline occurs andthe period of time over which that occurs appear to be the fundamentalcausative factors. Accordingly, the inventors had the insight thatcombining a GLP-1 antagonist, such as exendin(9-39), with anamylinomimetic, such as an amylin peptide or another agent that delaysgastric emptying, can improve the treatment of hyperinsulinemichypoglycemia, at least for some patients, relative to treatment with aGLP1A alone.

The novel formulations that result from combining a lyophilized amylinpeptide with exendin(9-39) or other GLP-1 antagonist in a compositionfor subcutaneous injection or inhaled pulmonary delivery provided by theinvention are particularly convenient for achieving simultaneousadministration of these agents in accordance with the invention. Forexample, one such formulation is prepared by dissolving amylin inaqueous solution together with exendin(9-39). This solution can be spraydried to produce a powder product of the invention. Alternatively theamylin peptide and the Exendin(9-39) product can be administered in theform of two separate powders that are co-administered using a devicewith a double chamber design, or alternatively administered as aphysical mixture of the two powders. Various other methods andformulations of the invention are prepared by combinations ofexendin(9-39) with a different agent that has the effect of delayedgastric emptying, such as an aluminum hydroxide antacid, any H2 ReceptorAntagonist (e.g. Ranitidine, Cimetidine, or Famotidine), or any ProtonPump Inhibitor, (e.g. Omeprazole, Lansoprazole, or Pantoprazole).

3.7 Dose Escalation

While the results in the examples below clearly demonstrate that dosesof 10, 20, and 30 mg in a simple immediate release formulation of theinvention will result in a therapeutic treatment effect (with desiredtreatment outcomes as above described), those practiced in the art willappreciate that other doses, as described herein, can be therapeuticallybeneficial for some patients. Some physicians may desire to treat with alow or initiating (starting) dose (e.g., 5-7.5 mg), escalate to anincreased if the initiating dose does not result in acceptable glycemiccontrol, and maintain the initiating dose if glycemic control issufficient. For example, some physicians may desire to treat with a lowor initiating (starting) dose and then escalate, e.g., the low and/orinitiating dose may be 5-10 mg, the middle dose may be 10-20 mg, and thehigh dose may be 20-30 mg, with the physician stopping the doseelevation when satisfactory (maximal) therapeutic benefit is obtained.

In some embodiments, a starting dose of 10 mg of GLP1A (e.g.,exendin(9-39)) in a morning dose and 10 mg GLP1A (e.g., exendin(9-39))in an evening dose is administered to the subject. If this dose does notresult in sufficient coverage in the morning (e.g., does not result insufficient glycemic control at the time of the morning meal), theevening dose may be increased, e.g., to 15 mg exendin(9-39) as theevening dose. In some embodiments, a starting dose of 15 mgexendin(9-39) in a morning dose and 15 mg exendin(9-39) in an eveningdose is administered to the subject. If this dose does not result insufficient coverage in the morning, the evening dose may be increased,e.g., to 20 mg exendin(9-39) as the evening dose.

Furthermore, depending upon the severity of the presenting patients'condition, for instance for patients presenting with a history ofseizure, loss of consciousness, or other neuroglycopenic episodes, ahigher and/or a more dilute dose of exendin(9-39) (or bioequivalentamounts of another GLP1A) with multiple injections optionally prescribedby the treating physician. For most patients, however, the treatingphysician will instruct the treated patient to follow standardprescribing information. An illustrative, non-limiting example of suchinstructions, based on use of the formulations as described in theexamples, is provided for illustrative purposes, as follows:

DOSAGE AND ADMINISTRATION: Inject subcutaneously in the abdomen, thigh,or upper arm. Administer within 60-150 minutes prior to morning andevening meals, or before the two main meals of the day, approximately6-10 hours apart. Initiate at a dose of 5 mg. If this dose does notresult in acceptable glycemic control, the dose can be increased to 10mg. If this does not result in acceptable glycemic control, the dose canbe increased stepwise to 15 mg (1×5 mg+1×10 mg dose), then 25 mg (1×15mg dose+1×5 mg dose) if needed.

DOSAGE FORMS AND STRENGTHS: Exendin(9-39) is supplied as a 1 to 5 mLdual chamber pen—either a disposable pen or one that reloads disposablecartridges—that delivers doses of: 5 mg per dose, 10 mg per dose, 15 mgper dose, or 25 mg per dose if needed. Contraindications, warnings, andprecautions might include, for example, a history of severehypersensitivity to a GLP-1 agonist (exenatide, for example) or GLP-1antagonist (exendin(9-39), for example). Hyperglycemia: May occur whenexendin(9-39), in particular if exendin(9-39) is used in connection withother medications known to raise plasma glucose (e.g., octreotide,diazoxide, corticosteroids). Renal Impairment: Exendin(9-39) is excretedrenally and should not be used in patients with severe renal impairmentor end-stage renal disease. Caution should be used in patients withrenal transplantation and when initiating or escalating doses inpatients with renal impairment. Should hypersensitivity reactions occur,(e.g., anaphylactic reactions and angioedema) the patient shoulddiscontinue exendin(9-39) and other suspect medications and seek medicaladvice. The label above is for an immediate or extended releaseformulation of exendin(9-39) administered by dual- or single-chamberpen—either disposable or re-loadable with cartridges—or by vial/syringedevice.

These and other benefits of the invention will be appreciated in greaterdepth upon contemplation of the examples below and accompanying figures,which conclusively demonstrate that administration of a GLP1A asdescribed herein can provide immediate and significant benefit topost-bariatric patients suffering hypoglycemic excursions afterconsuming normal amounts of glucose. Based upon American Society ofMetabolic and Bariatric Surgery (ASMBS) recommendations (seeasmbs.org/patients/life-after-bariatric-surgery), post-bariatric surgerypatients are encouraged to limit their carbohydrate intake to 50 gramsper day or less. Patients in the examples provided were administered 75grams of carbohydrate within a 20 minute period of time, amounting to1.5-fold the total ASMBS recommended daily intake. Thus based upon thesuccess demonstrated in the examples wherein administration of a GLP1Aprevented hypoglycemia and markedly improved symptoms after a highcarbohydrate load, under ordinary conditions, similar or greaterefficacy would be anticipated.

4. Examples 4.1 Example 1 Continuous IV Infusion of Exendin(9-39)Effectively Reverses Hyperinsulinemic Hypoglycemia and AssociatedSymptoms

A randomized placebo-controlled blinded cross-over Phase 1 study wasconducted to determine whether continuous IV infusion of exendin(9-39)can effectively reverse hyperinsulinemic hypoglycemia and associatedsymptoms. Exendin(9-39) was acquired as a lyophilized peptide:exendin(9-39) acetate 10 mg/vial from Bachem (Clinalfa, Laufelfingen,Switzerland). For preparation of the IV infusate, lyophilizedexendin(9-39) was solubilized with 20 ml 0.9% normal saline (NS) forevery 10 mg peptide, then diluted in 100 ml 0.9% NS and 50 ml of 25%human serum albumin, in a PVC-free, DEHP-free 1 L infusion bag. The bagwas covered with an opaque IV bag cover to aid with blinding. Anidentical-appearing bag was prepared, constituting the placebo infusate,containing the same volume of infusate (NS only) without the presence ofpeptide or albumin. Eight patients with hyperinsulinemic hypoglycemiawere randomized to receive an infusion of placebo and an infusion ofexendin(9-39) in cross-over design during an oral glucose tolerance test(OGTT) on two separate days separated by no greater than 2 weeks.Patients were asked to fast for 12 hours prior to the infusion of studydrug or placebo, and infusions and OGTTs were carried out the Center forTranslational Research Unit (CTRU) at Stanford University. On the day ofadmission to CTRU, 2 IV lines were placed for infusion of study drug andblood collection. Fasting blood was drawn at T-40 minutes. At T-30minutes, an IV bolus of 7,500 pmol/kg exendin(9-39) or placebo wasadministered over 1 minute, while a continuous IV infusion ofexendin(9-39) at a rate of 500 pmol/kg/min (providing an infusion doseof about 0.35 mg/kg) or placebo (0.9% saline) was initiated and run for210 minutes. At T+0 minutes an OGTT was initiated, wherein patients wereinstructed to consume a 75 g glucola drink over 20 minutes.

Plasma samples were collected at T−40, T+0, T+30, T+45, T+60, T+90,T+105, T+120, T+150, T+180 and at each timepoint immediately taken tothe laboratory for processing. The following assays were then conducted:glucose, insulin, GLP-1, GIP, glucagon, and exendin(9-39). If glucoselevels dropped to 50 mg/dL or less, the test was stopped andinvestigators intervened as needed to normalize glucose. At T−40 andconcomitant with timed blood draws, a graded symptom questionnaire wascompleted repetitively by patients. This questionnaire was adapted fromtwo validated hypoglycemia assessment tools, by segregating symptomsinto three clear factors: autonomic, neuroglycopenic, and malaise, andthen by adding a severity gradation scale, such that patients rated theseverity of each reported symptoms from 1-5 (1: least severe; 5: mostsevere).

As shown in FIG. 1B and Table 1, patients exhibited an average glucosenadir of approximately 80 mg/dL during exendin(9-39) infusion, ascompared to a nadir of <50 mg/dL during placebo infusion. Patients alsoexhibited a marked decrease in plasma insulin concentrations duringexendin(9-39) infusion (see FIG. 1A and Table 1).

Metabolic responses, including plasma GLP-1, GIP, and glucagonresponses, were measured as shown in FIG. 2 A-C, and Table 2. Althougharea under the curve (AUC) values were calculated as shown in Tables 1and 2, the presentation of the data graphically, as presented in FIGS. 1and 2, is more informative because subject OGTTs were stoppedprematurely if they became hypoglycemic (as they did in 100% of casesduring placebo infusion). For calculation of AUC in cases of prematurecessation of the OGTT, the last value was carried forward. Patients werealso assessed for hypoglycemic symptoms during exendin(9-39) infusionvs. placebo infusion. As shown in FIG. 3, continuous exendin(9-39)infusion substantially improved symptoms of hypoglycemia, asdemonstrated by the dramatically reduced total hypoglycemic symptomassessment score. Additionally, to isolate symptoms associated withglucose rise and fall, two subscores were included: the “Glucose Fall”score, which encompasses symptoms associated with the fall in glucose tonadir, and the “Glucose Rise” score, which encompasses symptomsassociated with the rise in glucose to peak.

The results demonstrate that continuous IV infusion of exendin(9-39)effectively reverses hyperinsulinemic hypoglycemia and associatedsymptoms.

4.2 Example 2 Single IV Bolus Injection of Exendin(9-39) ReversesHypoglycemia Only if Timed Coincide with Peak GLP-1 PlasmaConcentrations

A trial was performed to assess whether a single bolus dose ofexendin(9-39) was able to prevent hypoglycemia in a 75 gram OGTT withsubjects with hyperinsulinemic hypoglycemia. Two subjects withhyperinsulinemic hypoglycemia were admitted to the research clinic aftera 12 hour overnight fast. An IV bolus of 7,500 pmol/kg exendin(9-39) wasprepared as in Example 1. The subjects consumed a 75 gram glucola atT=0. GLP-1 levels are predicted to peak about 60 min after theadministration of glucola (see, Myint et al., European Journal ofEndocrinology, 2012, 166:951-955; see also FIG. 4D). After consuming theglucola, the subjects were infused intravenously with an IV bolus ofexendin(9-39) over 1 minute, with the timing of the IV bolusadministration relative to the 75 gram OGTT altered on different days,as follows: T=0, T+20, and T+50. Plasma was assayed at T−40, T+0, T+30,T+45, T+60, T+90, T+105, T+120, T+150, T+180 and at each timepointimmediately taken to the laboratory for processing. Measurements weretaken for glucose, insulin, GLP-1, GIP, glucagon, and exendin(9-39).Bioavailability/PK profile of IV exendin(9-39) was evaluated by Cmax,Tmax, AUC0-∞, AUClast, VZ, CL, and T_(1/2). Exendin(9-39) concentrationwas measured by radioimmunoassay (RIA) as described in Kielgast et al.,Diabetes, 2011, 60:1599-1607.

As shown in FIGS. 4A-C, dosing of the IV bolus of exendin(9-39) at 0minutes or 20 minutes following administration of glucola did notprevent hypoglycemia, whereas dosing at 50 minutes after administrationof glucola did prevent hypoglycemia. See, figure legend. FIGS. 4A-Ddemonstrates that peak plasma exendin(9-39) concentrations in the rangeof 500-600 nMol/L by radioimmunoassay at the time of peak plasma GLP-1concentrations are required to avoid a glucose nadir below 50 mg/dL. Theresults shown in FIG. 4A-D suggest that in the absence of continuous IVinfusion, or in the absence of an IV bolus timed precisely to the peakpredicted GLP-1 plasma concentrations, hypoglycemia cannot be averted.

Exendin(9-39) plasma levels can be measured using a radioimmunoassay(RIA) generally as described in Kielgast et al., Diabetes, 2011,60:159-1607. Exendin(9-39) plasma levels can be measured using liquidchromatography-mass spectrometry (LCMS) methodology generally asdescribed in Lasaosa et al., J. Chromatogr B Analyt Technol Biomed LifeSci, 2014, 0:186-191. We refer to both methods in the discussion herein,and both methods are used in the scientific literature. We observed thatmeasurement of plasma exendin(9-39) values using RIA were significantlyhigher than values determined using LCMS. We believe the LCMS values aremore accurate. For definitional purposes, a claimed exendin(9-39)concentration (e.g., Cmax) refers to the absolute quantity ofExendin(9-39) which may be determined by LCMS or another equallyquantitative method.

FIG. 5A depicts an average of eight patients' plasma exendin(9-39)concentrations at various timepoints following a 7,500 pmol/kg IV bolusof exendin(9-39) at T-30 minutes, followed by continuous IV fusion at arate of 500 pmol/kg/min over 210 minutes as described in Example 1. Seegraph line with error bars. It has also been reported that in healthysubjects an intravenous infusion of exendin(9-39) at 500 pmol/kg/minfully reverses the glucose lowering effect of GLP-1. See, Edwards etal., Diabetes, 1999, 48:86-93. Based on the measured plasmaexendin(9-39) concentrations as shown in FIG. 5A, a steady plasmaexendin(9-39) concentration of approximately 500 nmol/L (as measured byradioimmunoassay) or of approximately 140 nmol/L (as measured by LCMS)is presumed to be required for efficacy.

FIG. 5A also shows the projected exendin(9-39) plasma concentration thatwould be expected from administering a single IV bolus of 7,500 pmol/kgexendin(9-39) at T−30 minutes. As previously reported, the half-life ofa single dose of intravenously administered exendin(9-39) is about 33.5minutes (see, Edwards et al., Diabetes, 1999, 48:86-93). Extrapolatingfrom the exendin(9-39) concentration measured at T=0 (about 300 nmol/Las measured by RIA) it was concluded that the exendin concentration atT=−30 is about 600 nmol/L given the half-life of intravenouslyadministered exendin(9-39). In view of the projected pharmacokineticresponse for exendin(9-39) and the time course of the development ofhypoglycemia following a meal (typically 1-3 hours after meals, withpeak GLP-1 levels expected at about 60 minutes after the meal), a singleIV bolus dose administered prior to or with a meal would likely not beeffective for treatment of hyperinsulinemic hypoglycemia, because theexendin(9-39) plasma concentration would be expected to be very low atthe predicted time of peak GLP-1 levels. Furthermore, even if an IVbolus having a higher dose of exendin(9-39) were administered, it wouldbe expected to exhibit similar pharmacokinetic properties of a shorthalf-life and rapid elimination from plasma. In view of the time coursefor the development of hypoglycemia and the lag between the time of ameal and the projected peak GLP-1 levels, even an IV bolus having ahigher dose of exendin(9-39) would not be expected to be efficacious inaverting hypoglycemia unless precisely timed with predicted peak plasmaGLP-1 levels.

4.3 Example 3 A Single Dose of Subcutaneously Injected Exendin(9-39)Effectively Reverses Hyperinsulinemic Hypoglycemia and AssociatedSymptoms

As described above in Example 1, it was found that an IV bolus of 7,500pmol/kg exendin(9-39) plus a continuous IV infusion of exendin(9-39) ata rate of 500 pmol/kg/min over 210 minutes was efficacious in reversinghyperinsulinemic hypoglycemia and associated symptoms. For the peptideexenatide, it has been reported that the absorption kinetics ofexenatide in rats most closely approximates human absorption kinetics.See, Chen et al., Interspecies Modeling Pharm Res., 2013, 30:751-760.Rat intravenous and subcutaneous dose escalation pharmacokinetic datapredicts that in humans, the Cmax of subcutaneously administeredexendin(9-39) would be substantially lower than the Cmax ofintravenously administered exendin(9-39). Accordingly, it was expectedthat a higher dose of exendin(9-39) would be needed for subcutaneousadministration, as compared to intravenous administration, in order forexendin(9-39), to be effective in reversing hyperinsulinemichypoglycemia.

To compare the pharmacokinetic parameters of intravenously orsubcutaneously administered exendin(9-39), a single IV bolus of 7,500pmol/kg exendin(9-39) or a single subcutaneous injection of 7,500pmol/kg exendin(9-39) was administered in one subject on two separatedays. The IV bolus consisted of 0.025 mg/kg of lyophilized exendin(9-39)(which equates to a dose of approximately 2 mg for an 80 kg patient)solubilized in 20 ml per 10 mg exendin(9-39) (approximately 4 ml normalsaline) and then diluted in 100 ml 0.9% normal saline for every 10 mgexendin(9-39) (approximately 20 ml 0.9% normal saline), to whichapproximately 10 ml 25% human serum albumin was added (50 ml 25% humanserum albumin for every 10 mg exendin(9-39)), for a total IV bolusinfusion volume of approximately 34 ml. The IV bolus infusion wasadministered over 1 minute. The subcutaneous injection consisted of0.025 mg/k of lyophilized exendin(9-39) (which equates to a dose ofapproximately 2 mg for an 80 kg patient) solubilized in 0.2 ml normalsaline and further diluted in 0.5 ml normal saline to a total volume of0.7 ml for subcutaneous injection in the arm. Plasma exendin(9-39)concentrations were measured by liquid chromatography-mass spectrometry(LCMS) as described in Lasaosa et al., supra Example 1. As shown in FIG.5B, the Cmax that was observed in subcutaneous administration ofexendin(9-39) was significantly lower than the Cmax observed inintravenous administration of exendin(9-39), further supporting thehypothesis that for subcutaneous administration, a higher dose ofexendin(9-39) would be required for preventing hyperinsulinemichypoglycemia, as compared to efficacious doses of intravenouslyadministered exendin(9-39).

A single ascending dose (SAD) study was performed to assess thepharmacokinetics, efficacy, and local tolerability of administeringexendin(9-39) by subcutaneous injection. For the SAD study, ninesubjects with hyperinsulinemic hypoglycemia were randomized to one offour experiments, each representing one of four subcutaneous doses ofexendin(9-39): 7,500 pmol/kg, 37,500 pmol/kg, 75,000 pmol/kg, or 112,500pmol/kg. Lyophilized exendin(9-39) acetate 10 mg/vial from Bachem(Clinalfa, Läufelfingen, Switzerland) was acquired for each experiment,with each 10 mg vial solubilized in 200 μl normal saline, then furtherdiluted with normal saline to a total dose of 7,500 pmol/kg, 37,500pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg (2.0 mg, 10 mg, 20 mg, or 30mg of exendin(9-39), respectively, based on a patient weight of 80 kg).The total volume of each injectate was held constant, with furtherdilution of injectate as required to result in a total volume ofinjectate of 0.7 ml. Of the nine subjects, five subjects were randomizedto receive one subcutaneous injection of 7,500 pmol/kg, 37,500 pmol/kg,75,000 pmol/kg, or 112,500 pmol/kg (2, 10, 20, and 30 mg, respectively,based on an 80 kg patient) in a volume of 0.7 ml normal saline, and foursubjects received two or more 0.7 ml injections of 75,000 pmol/kg, or112,500 pmol/kg in order to maintain an injectate concentration of about15 mg/ml or less.

Subjects fasted overnight for 12 hours and were admitted to the researchclinic. One IV line was placed in the patient for blood collection.Fasting blood was drawn. Subjects were injected subcutaneously in theabdomen with the dose of exendin(9-39) to which they were randomized,and were blinded as to which dose they were receiving. For the subjectsreceiving a dose of 37,500 pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg,an OGTT was initiated at T+0 minutes, wherein patients were instructedto consume a 75 g glucola drink over 20 minutes. Plasma samples werecollected at T−10, T−0, T+15, T+30, T+45, T+60, T+75, T+90, T+105,T+120, T+135, T+150, T+165, T+180, T+210, T+240, T+300, T+480, andT+1440, and at each timepoint the samples were immediately taken to thelaboratory for processing.

The following parameters were evaluated: 1) plasma glucose, insulin,glucagon, GLP-1, and GIP concentration; 2) bioavailability/PK profile ofsubcutaneous exendin(9-39): Cmax, Tmax, AUC0-∞, AUClast, VZ, CL,T_(1/2), and bioavailability; 3) local tolerability after subcutaneousinjection of exendin(9-39) utilizing a Visual Analog Scale (VAS) and aNumeric Rating Scale (NRS); and 4) local swelling as measured by caliperat timed intervals along the long and short axes of the swelling andbump height. Patients were also assessed for hypoglycemic symptoms usinga graded symptom questionnaire in which patients rated the severity ofspecifically recited hypoglycemia symptoms from 1-5 (1: least severe; 5:most severe) at specific timepoints, from which a “Glucose Rise” score,“Glucose Fall” score, and “All Timepoints” score were calculated.Exendin(9-39) concentration was measured by liquid chromatograph-massspectrometry as described by Lasaosa et al., supra Example 1.

For the 8 subjects who were administered a subcutaneous dose of 37,500pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg, the plasma glucoseconcentrations were measured during the OGTT. (The dose of 7,500 pmol/kgthat was administered to subject 1 was presumed to be subtherapeutic,and so an OGTT was not administered to this subject.) For the remainingeight subjects who were administered a subcutaneous dose ofexendin(9-39) and an OGTT, none of the subjects became hypoglycemicafter subcutaneous injection at doses ranging from 35,000-112,500pmol/kg. Thus, prevention of hypoglycemia was achieved at allsubcutaneous dose levels. In contrast, all of the subjects receivingplacebo became hypoglycemic during the OGTT. As shown in FIG. 6, theaverage plasma nadir for the 8 subjects administered a subcutaneous doseof exendin(9-39) was 78 mg/dL, versus <50 mg/dL for the placebo.Additionally, as shown in Table 3, the average subject symptomaticresponse was significantly improved for the subjects who wereadministered the subcutaneous dose of exendin(9-39) and OGTT, asmeasured by a dramatically reduced Overall Symptom Score (14.6 vs. 20.6for placebo) and Symptom Fall Score (4 vs. 22 for placebo).

As shown in Table 4, subcutaneous administration of exendin(9-39) as asingle injection at a dose ranging from 37,500 pmol/kg to 112,500pmol/kg (approximately 10-30 mg) and a constant volume of 0.7 ml wasefficacious for preventing hypoglycemia, for example as shown by theplasma glucose nadir. An injectate concentration of approximately 15mg/ml (the 37,500 pmol/kg dose) resulted in the greatest pharmacodynamicresponse, as defined by Cmax and dose-normalized Cmax. For the subjectswho were subcutaneously administered exendin(9-39) at relativelyequivalent concentrations (approximately 13-16 mg/ml), as shown in Table5, exendin(9-39) administration was efficacious in preventinghypoglycemia, for example as shown by the plasma glucose nadir. Forthese patients, Table 5 shows that there was an increasingly favorablePK response with increasing dose, as defined by Cmax and T_(1/2).

As shown in FIG. 8, a strong correlation was found between the percentincrease in plasma glucose nadir concentrations (comparing plasmaglucose nadir after subcutaneous injection of exendin(9-39) to baselineplasma glucose nadir) and the peak plasma exendin(9-39) concentrations(Cmax).

Surprisingly, the clinical efficacy achieved for the subcutaneouslyadministered doses of exendin(9-39) tested in this SAD study wasequivalent to the efficacy that was achieved by continuous IV infusionof larger quantities of exendin(9-39) as described in Example 1, asshown for example in Table 3 for the plasma glucose nadir, AUC glucose,and the Symptom Fall Score parameters.

In view of the efficacy of the subcutaneously administered dose levelsas demonstrated in this example, the efficacy, safety, tolerability, andpharmacokinetics of subcutaneous administration of exendin(9-39) over adefined time period is evaluated, for example, as described in Example 4and Example 5 below.

4.4 Example 4 Multi-Ascending Dose Trial to Assess the Efficacy,Tolerability, and Pharmacokinetic Profile of BID Exendin(9-39) inPatients with Post-Bariatric Hyperinsulinemic Hypoglycemia

This example describes a Phase 2a clinical study protocol for evaluatingthe safety, tolerability, efficacy, and pharmacokinetic profile of BIDexendin(9-39) administered subcutaneously over 3 days to patients withpost-bariatric hyperinsulinemic hypoglycemia.

TABLE B Study objectives and endpoints Objective Endpoint Primary: Toevaluate the Response rate in plasma glucose nadir, defined treatmenteffect as proportion of patients in each dose arm with on plasma glucoseno plasma glucose ≤50 mg/dL at any timepoint of SC BID Ex9 from 0-180minutes during OGTT on Day 3 of treatment vs. on Day 0. Secondary: Toevaluate the Improvement in composite symptom score as treatment effectcompared to baseline during OGTT on Day 3 of on symptoms of treatmentvs. on Day 0. hypoglycemia of SC BID Ex9 To assess the pharma- Plasma PKparameters include AUC_(0-12 h), C_(max), cokinetics of SC BID T_(max),T_(1/2), and C_(trough), after SC injection. Ex9 at each dose level Toassess the safety AEs, laboratory parameters, vital signs; NMR andtolerability of score, VAS score. SC BID Ex9 at each dose level

Overview: The study is a single-blinded, dose-randomized, cross-overdesign study that is being conducted at the Stanford University Schoolof Medicine. All subject visits will take place in the Clinical andTranslational Research Unit (CTRU). Sixteen to twenty eligible subjectswill be assigned to one of five dose levels (2.5 mg, 5 mg, 10 mg, 15 mg,20 mg) to receive subcutaneous injection of BID exendin(9-39)administered for three days. After a baseline Oral Glucose ToleranceTest (OGTT) is conducted on Day 0 wherein metabolic and symptomaticanalyses will occur, subjects will return to the research clinic on Day1 to initiate a BID dosing schedule for 3 days. During this time,subjects will return daily for fasting labs in the morning, a morningdose, PK sampling, and an evening trough sample, followed immediately bythe second daily dose at T+720 min. Safety, tolerability, andpharmacokinetic parameters will be measured on a daily basis for thefull three day duration of the study, after which a repeat OGTT isperformed on the morning of Day 3 after the morning dose to evaluate forefficacy (no hypoglycemia and reduction in composite symptom score). Day4 will consist solely of clinical safety monitoring with a plasma troughdrawn 1440 minutes after the last Day 3 injection. This study ifproperly conducted is expected to demonstrate that BID dosing can resultin meaningful therapeutic activity in each dosing arm. See, FIG. 9.

Randomization/blinding: For the first four subjects dosed, the subjectswere randomly assigned to one of the following dose levels: 2.5 mg, 5mg, or 10 mg. The remaining subjects will be randomly assigned to one ofthe following dose levels: 10 mg, 15 mg, or 20 mg. All subjects willremain blinded throughout. With the exception of the PI andsub-investigator who will remain un-blinded for safety purposes, allsite personnel including nurses and study coordinators, who conductpatient symptom surveys, will remain masked to treatment assignment.

Study drug preparation and dispensation: All doses will be prepared to atotal concentration of ≤15 mg/ml of exendin(9-39) in normal saline. Each10 mg vial of lyophilized exendin(9-39) will be diluted in either 1 mlnormal saline if a 10 mg/ml concentration is administered, or 0.7 mlnormal saline if a 14 mg/ml concentration is administered. For dosesrequiring total volume of injection >1 ml, 2 injections will beemployed.

Oral Glucose Tolerance Test (OGTT): The OGTT will consist ofadministration of one 75 mg gram glucola drink with 1 gram of crushedacetaminophen to be consumed over 20 minutes.

Assays: Metabolic: glucose, c-peptide, insulin, GLP-1, GIP, glucagon;PK: AUC₀₋₇₂₀, C_(max), T_(max), T_(1/2), C_(trough).

Anticipated PK profile: It was anticipated, based on the prior resultsfor a single subcutaneous injection (as shown in Example 3), that afteradministration of a 5 mg, 10 mg, 15 mg, or 20 mg dose the plasmaconcentration of exendin(9-39) would return to <20 ng/mL or even closeto 0 ng/mL within 720 minutes of injection. However, based on theintermediate results of BID dosing for 3 days as shown in FIG. 10 and asdiscussed below, it is expected that administration of a 10-30 mg dosewill result in a higher nadir, such as a nadir of about 30-80 ng/mlwithin 720 minutes after injection.

It is expected that a dosage of 5 mg BID, 10 mg, 15 mg or 20 mg BIDexendin(9-39) will demonstrate a therapeutic benefit for one or morepatients in the 3-day trial. A “therapeutic benefit” may be defined withreference to effect on plasma glucose. For example, in some instances adosage of exendin(9-39) provides a therapeutic benefit for a patientwhen the patient has no plasma glucose ≤50 mg/dL at any timepoint from0-180 minutes during OGTT on Day 3 of treatment as compared to Day 0. Insome instances a dosage of exendin(9-39) provides a therapeutic benefitfor a patient when the patient has at least a 15% increase in plasmaglucose nadir during OGTT on Day 3 relative to Day 0. In some instancesa dosage of exendin(9-39) provides a therapeutic benefit for a patientwhen the patient has at least a 15% increase in AUC glucose. In someinstances a dosage of exendin(9-39) provides a therapeutic benefit for apatient when the patient has a statistically significant decrease in theseverity of one or more symptoms of hypoglycemia overall during the OGTTand/or of neuroglycopenic symptoms elicited during the glucose “Fall”period of the OGTT relative to Day 0. In some instances a dosage ofexendin(9-39) provides a therapeutic benefit for a patient having aplasma glucose nadir ≤50 mg/dL at baseline when the patient exhibits aplasma glucose nadir ≥55 mg/dL after a defined treatment period (e.g.,after a 3 day treatment period). Intermediate results: Four subjectswere randomized to one of three dose levels (2.5 mg, 5 mg, or 10 mg) toreceive subcutaneous injection of BID exendin(9-39) administered forthree days.

Patient 1 was administered a dose of 5 mg at a concentration of 10 mg in1 ml, subcutaneously administered in the abdomen. For Patient 1, a 13.1%increase in AUC glucose was observed as compared to baseline, buthypoglycemia was not prevented, as defined by plasma glucose ≤50 mg/dL.Patient 2 was administered a dose of 2.5 mg at a concentration of 10 mgin 1 ml, subcutaneously administered in the abdomen. For Patient 2, an8.8% increase in AUC glucose was observed, but hypoglycemia was notprevented, as defined by plasma glucose ≤50 mg/dL. Patient 3 wasadministered a dose of 5 mg at a concentration of 10 mg in 1 ml,subcutaneously administered in the arm. For Patient 3, a 16.3% increasein AUC glucose was observed, but hypoglycemia was not prevented, asdefined by plasma glucose ≤50 mg/dL.

Patient 4 was administered a dose of 10 mg at a concentration of 10 mgin 1 ml, subcutaneously administered in the arm. For this patient,hypoglycemia was not prevented, as defined by plasma glucose ≤50 mg/dL.

These intermediate pharmacodynamic results demonstrate an increasingtherapeutic benefit, as defined by % increase in glucose AUC withincreasing doses administered, with one of the two patients dosed with 5mg experiencing a greater than 15% increase in AUC glucose as comparedto AUC glucose during a baseline oral glucose tolerance test. Whilehypoglycemia as defined by plasma glucose ≤50 mg/dL was not prevented, atherapeutic dose response was achieved, illustrating that doses of 10-30mg will result in improved glycemic control, as further shown by Example3 and FIG. 8.

The pharmacokinetic parameters obtained from the 3-day BID dosing ofPatents 1-4 are shown in Table 6 below. The single subject dosed at 10mg for 3 days was severely disabled, experiencing daily episodes ofsymptomatic neuroglycopenia, and requiring placement of gastrostomy tubeinto the remnant stomach for route of nutrient administration. Theseintermediate results demonstrate that total exendin(9-39) exposureincreases with increasing dose. As shown in Table 6, AUC was increasedby about 1.5-1.7-fold, and Cmax was increased by about 50% with a 5 mgdose escalation from 5 mg to 10 mg. Similar degrees of increase areexpected to be observed for AUC and Cmax with an escalation to a 15 mgdose. For a 15 mg dose, a Cmax value is expected to be in thetherapeutically effective range of approximately 150-200 ng/ml. Interimpharmacokinetic results from this 3-day trial also demonstrate that onaverage, AUC plasma concentrations increase with increasing days of BIDdosing. A higher trough was observed at Day 3 than at Day 1, suggestingseveral days (e.g., 3-5 days) may be required to reach steady state.Thus, the results of this study support efficacy of the 15 mg dose atDay 3 of treatment. The results of this study also support efficacy ofthe 10 mg dose in less severely disabled patients and/or with longer(e.g., 5 days) treatment.

Comparison of the pharmacokinetic responses to abdominal versus arminjection of 5 mg doses in patient 1 and patient 3 demonstrates that aquicker absorption profile with higher exposure as defined by Cmax (seeTable 6), can be achieved by administration of the injectate into anarea with less subcutaneous fat, as may be the abdominal subcutaneousarea after bariatric surgery weight loss. A slower absorption profilewith longer exposure, as defined by AUClast (see Table 6) can beachieved by administration of the same dose into an area with relativelymore subcutaneous fat, such as the arm area may have after bariatricsurgery weight loss.

4.5 Example 5 Multiple Doses of Subcutaneously Injected Exendin(9-39)Safely and Effectively Reverse Hyperinsulinemic Hypoglycemia

This example demonstrates the method of the invention in which amulti-site multi-ascending dose (MAD) format is used to evaluate theefficacy, safety, and pharmacokinetics of a 28-day trial of immediaterelease subcutaneous exendin(9-39) administered BID in patients withsevere post-bariatric hypoglycemia. The primary objective of this trialis to demonstrate the efficacy of exendin(9-39) on plasma glucose levelsduring a 3-hour oral Glucose Tolerance Test (OGTT) at the end of 4-weektreatment. This trial is also intended to demonstrate the efficacy ofexendin(9-39) on the frequency and severity of hypoglycemia incidenceand associated symptoms in patients with severe post-bariatrichypoglycemia. This trial also demonstrates the pharmacokinetics andpharmacodynamics of exendin(9-39) at each dose level. Furthermore, thistrial demonstrates the safety and tolerability profile of the immediaterelease subcutaneous formulation of exendin(9-39) in patients withsevere post-bariatric hypoglycemia.

This is a multi-center, double-blind, randomized, placebo-controlled,parallel-group, two exendin(9-39) dose levels, phase 2 study in patientswith severe post-bariatric hypoglycemia. Approximately 36 patients willbe recruited. Eligible patients will have a confirmed diagnosis ofsevere hypoglycemia post-bariatric via Whipple's triad and OGTT. Thestudy is divided into three phases, as follows:

Screening phase: All potential subjects will complete an oral glucosetolerance test (OGTT), wherein if plasma glucose falls to less than orequal to 60 mg/dL and all other eligibility criteria are met, thepatient will be allowed to enroll in the study. In cases of out of rangelaboratory values, with the exception of laboratory tests related tore-feeding syndrome, subjects are permitted to re-screen one time.

4-week randomized treatment (RT) period: All enrolled subjects willparticipate in a 4-week randomized treatment period wherein subjectswill be randomized to one of two exendin(9-39) doses (e.g., 10 mg and 20mg, 10 mg and 15 mg, or 15 mg and 20 mg) administered BID or matchingplacebo of the 2 doses. The ratio of treatment assignment to the firstexendin(9-39) BID dose, the second exendin(9-39) BID dose, the firstmatching placebo dose, and the second matching placebo dose will be2:2:1:1. During the RT period, the subjects will undergo continuousglucose monitoring wearing Dexcoms at home.

Open-label extension (OLE) period: All patients completing Week 4 of therandomized treatment period and experiencing benefit with exendin(9-39)at the end of RT will be eligible to enter the OLE period. During theOLE period, the dose administered will either be an optimal fixed doselevel selected at the end of the randomized treatment period of thestudy or up-titrated to 20 mg BID until any of the following occur:completed 12 months of the open-label extension; unacceptable toxicity;lack of efficacy; protocol deviation; patient withdrew consent; lost tofollow-up; death; and study discontinues per the sponsor.

The primary efficacy endpoint is measured as the response rate in plasmaglucose level at the end of the 4-week RT, defined as the proportion ofpatients either (1) without plasma glucose ≤55 mg/dL for patients whoseglucose nadir is ≤50 mg/dL at baseline OGTT; or (2) without plasmaglucose ≤60 mg/dL for patients whose glucose nadir is 55−≤60 mg/dL atbaseline OGTT. Secondary efficacy endpoints are measured as theimprovement in neuroglycopenic symptom score during OGTT at the end ofRT (Week 4), where neuroglycopenic symptoms include inability toconcentrate, confusion, weakness, drowsiness, dizziness, blurred vision,difficulty speaking (modified from the Edinburgh Hypoglycemia Score,Hepburn 1991); the proportion of patients with severe hypoglycemiaduring the 4-week RT, where severe hypoglycemia is defined an eventrequiring assistance of another person to actively administercarbohydrates, glucagon, or take other corrective actions with a bloodglucose concentration of <50 mg/dL by continuous glucose monitoring(CGM); the proportion of patients with any hypoglycemia event betweenWeek 2 and Week 4, where hypoglycemia is defined as a plasma glucoseconcentration of ≤55 mg/dL by continuous glucose monitoring (CGM)[Hypoglycemia after Roux-En-Y gastric bypass: detection rates ofcontinuous glucose monitoring (CGM) versus mixed meal test Kefurt 2014];and the Change in Quality of life at Week 4 from baseline as evaluatedusing Short-Form 36 (SF-36) domain scores.

The pharmacokinetic and pharmacodynamics endpoints to be measuredinclude C_(max), T_(max), T_(1/2), C_(trough), AUC of exendin(9-39).Exploratory endpoints will include insulin (AUC, Peak, ISR, ICR),GLP-1/GIP, and glucagon concentrations.

4.6 Example 6 Diminished Novelty Preference Scores on the Visual PairedComparison Test in Patients with Hyperinsulinemic Hypoglycemia asCompared to Insulin Sensitive Healthy Controls Predicts Mild CognitiveImpairment Indicative of Cumulative Hyperinsulinemic HypoglycemiaEffects

This example describes one embodiment of the method of invention whichis conducted as a pilot study to evaluate whether patients withhyperinsulinemic hypoglycemic exhibit early signs of amnestic mildcognitive impairment (aMCI) in comparison to health controls, andtherein may benefit from GLP1A for prevention of further cognitivedecline. In this design, six patients with hyperinsulinemic hypoglycemiaand six sex- and age-matched insulin sensitive controls are enrolled.Participants arrive at the research clinic fasted for 12 hours. They areseated in front of a monitor, with eye position calibrated for eachparticipant using a 9-point array. System parameters are adjusted untilthe participant's fixations accurately mapped onto the calibrationpoints. Participants are told that images will appear on the computerscreen and are instructed to look at the images “as if watchingtelevision.” During testing, the participants eye fixations and eyemovements are recorded throughout the 2 phases of the Visual PairedComparison (VPC) test; a familiarization phase followed by a test phase.During the familiarization phase, 2 identical images are presented sideby side on the monitor for 5 seconds. The monitor then goes dark for adelay interval of 2 seconds. In the test phase, 2 images are againpresented side by side for 5 seconds. One of the images is identical tothe image presented during the familiarization phase and the other was anovel image. Presentation of the novel image on the left or right sideis selected pseudo randomly and distributed equally.

Eye fixation and movement data for each participant are extracted andanalyzed, with fixation defined as a point of gaze continually remainingfor a period of 100 milliseconds or more looking at the area of eitherthe novel image or the area of the familiar image. Eye data arecharacterized using percent looking time on the novel image, shown inFIG. 11 as “Novelty Preference Score.” As shown in FIG. 11, patientswith hyperinsulinemic hypoglycemia scored lower than matched controls,and one can extrapolate that this condition may confer higher risk ofconversion to aMCI or Alzheimer's disease than healthy controls. Giventhat hyperinsulinemic hypoglycemia is a lifelong condition, and patientswith this condition typically have multiple episodes of hypoglycemia perweek, the cumulative effects on cognition may be substantial. Preventionof such episodes with the method of the invention could havelong-ranging effects, such as prevention of cognitive decline.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

TABLE 1 Subject metabolic responses to OGTT with and without intravenousinfusion of Exendin (9-39) vs. non-surgical controls HH Placebo^(a) HHEx(9-39)^(a) Non-Surg controls^(a) P-value^(b) P-value^(c) P-value^(d)(n = 8) (n = 8) Fasting plasma glucose (mg*dl⁻¹) 91.8 ± 1.2 94.7 ± 3.9100.6 ± 4.3  0.414 0.06  0.322 Peak postprandial glucose (mg*dl⁻¹) 235.4± 11.0 225.5 ± 15.1 152.3 ± 6.1  0.432 0.000 0.001 Time to peak glucose(min) 56.3 ± 3.8 37.5 ± 4.9 45.0 ± 5.7 0.011 0.120 0.334 Nadirpostprandial glucose (mg*dl⁻¹) 46.1 ± 1.9 78.7 ± 5.4 74.9 ± 3.8 0.0000.000 0.570 Time to hypoglycemia (min) 137.5 ± 5.3  NA NA NA NA NA Deltapeak-nadir glucose (mg*dl⁻¹) 189.3 ± 10.1 146.8 ± 15.3 77.4 ± 7.6 0.0120.000 0.001 Rate of glucose decline (mg*dl⁻¹*min⁻¹)  3.1 ± 0.5  1.1 ±0.3  0.5 ± 0.2 0.001 0.001 0.189 AUC glucose (0, 60)(mg*dl⁻¹*min⁻¹)10402.5 ± 309.3  10905.9 ± 624.1  8120.6 ± 287.1 0.278 0.000 0.001 AUCglucose(60, 180)(mg*dl⁻¹*min⁻¹) 11318.9 ± 573.3  15397.5 ± 1180.713346.3 ± 504.9  0.010 0.019 0.378 AUC glucose(0, 180)(mg*dl⁻¹*min⁻¹)21721.4 ± 701.8  26303.4 ± 1785.8 21466.9 ± 642.2  0.020 0.793 0.023Fasting plasma insulin (uU*ml⁻¹)  4.0 ± 19.2  3.2 ± 0.8 15.0 ± 1.2 0.2600.000 0.000 Peak postprandial insulin (uU*ml⁻¹) 200.3 ± 28.5  88.3 ±23.0 86.0 ± 8.3 0.000 0.002 0.928 Time to peak insulin (min) 60.0 ± 5.748.8 ± 5.5  67.5 ± 12.4 0.285 0.590 0.187 AUC insulin (0,60)(uU*ml⁻¹*min⁻¹) 6220.3 ± 766.4 3368.9 ± 832.4 3420.0 ± 375.7 0.0010.005 0.956 AUC insulin (60, 180)(uU*ml⁻¹*min⁻¹) 4591.6 ± 876.3 2462.2 ±524.8 6532.5 ± 607.9 0.038 0.090 0.000 AUC insulin (0,180)(uU*ml⁻¹*min⁻¹) 13605.5 ± 1819.0  5831.1 ± 1281.0 9952.5 ± 869.90.001 0.092 0.019 Insulin at glucose <55 mg/dl (uU*ml⁻¹) 17.5 ± 4.7 NANA NA NA NA Insulinogenic Index (0, 30)  1.2 ± 0.2  0.7 ± 0.2  1.2 ± 0.40.001 0.927 0.286 Insulinogenic Index (0, 60)  1.4 ± 0.2  0.6 ± 0.1  1.9± 0.4 0.000 0.239 0.006 ^(a)Data are presented as mean ± SEM^(b-d)P-value by two-sided student's t-test ^(b)P-value HH Placebo vs.HH Ex(9-39) ^(c)P-value HH Placebo vs. Non-Surg Controls ^(d)P-value HHEx(9-39) vs. Non-Surg Controls

TABLE 2 Subject incretin responses to OGTT with and without intravenousinfusion of Exendin (9-39) HH Placebo^(a) HH Ex(9-39)^(a) P-value^(b) (n= 8) Fasting GLP-1 (pmol*L⁻¹)  9.0 ± 0.4  9.4 ± 1.2 0.191 Peak GLP-1(pmol*L⁻¹) 86.0 ± 6.1  82.3 ± 13.9 0.857 Time to peak GLP-1 (min) 42.9 ±6.1 38.6 ± 5.5 0.604 AUC GLP-1 (0, 60)(pmol*L⁻¹*min⁻¹) 3270.0 ± 214.13267.9 ± 606.8 0.998 AUC GLP-1 (60, 180)(pmol*L⁻¹*min⁻¹) 3135.7 ± 265.73621.4 ± 416.0 0.429 AUC GLP-1 (0, 180)(pmol*L⁻¹*min⁻¹) 6405.7 ± 423.06889.3 ± 941.2 0.700 Peak Insulin to Peak GLP-1 ratio  2.6 ± 0.1  1.3 ±0.4 0.059 Fasting GIP (pmol*L⁻¹*min⁻¹) 14.4 ± 1.4 14.0 ± 1.5 0.824 PeakGIP (pmol*L⁻¹*min⁻¹)  93.1 ± 12.8  86.6 ± 12.0 0.005 Time to peak GIP(min) 42.9 ± 6.0 34.3 ± 4.3 0.356 AUC GIP (0, 60)(pmol*L⁻¹*min⁻¹) 3831.4± 495.9 3267.9 ± 606.8 0.127 AUC GIP (60, 180)(pmol*L⁻¹*min⁻¹) 3722.1 ±168.9 3165.0 ± 396.9 0.084 AUC GIP (0, 180)(pmol*L⁻¹*min⁻¹) 7553.6 ±617.9 6420.0 ± 941.2 0.003 Peak Insulin to Peak GIP ratio 2.4 0.4 1.30.4 0.000 Fasting glucagon (pmol*L⁻¹) 40.5 ± 2.8 41.9 ± 2.9 0.567 Peakglucagon (pmol*L⁻¹) 82.8 ± 6.7 92.3 ± 6.4 0.079 Time to peak glucagon(min)  60.0 ± 12.7  45.0 ± 11.3 0.470 AUC glucagon (0,60)(pmol*L⁻¹*min⁻¹) 3796.6 ± 306.2 4430.5 ± 285.1 0.033 AUC glucagon(60, 180)(pmol*L⁻¹*min⁻¹) 7999.8 ± 912.2 8017.5 ± 696.3 0.981 AUCglucagon (0, 180)(pmol*L⁻¹*min⁻¹) 11584.4 ± 1252.9 12019.3 ± 941.9 0.981

TABLE 3 Placebo^(a) SC Ex(9-39)^(a) IV Ex(9-39)^(a) P-value^(b)P-value^(c) Subject metabolic response (n = 8) (n = 8) (n = 8) Fastingplasma glucose (mg*dl⁻¹) 91.6 ± 1.7 94.5 ± 1.7 94.7 ± 3.9 0.125 0.966Peak postprandial glucose (mg*dl⁻¹) 229.3 ± 13.2 252.3 ± 23.7 225.5 ±15.1 0.258 0.358 Time to peak glucose (min) 54.5 ± 3.7 52.5 ± 4.9 37.5 ±4.9 0.351 0.049 Nadir postprandial glucose (mg*dl⁻¹) 47.7 ± 1.6 77.9 ±4.1 78.7 ± 5.4 <.001 0.906 Time to hypoglycemia (min) 135.5 ± 4.9  NA NANA NA Delta peak-nadir glucose (mg*dl⁻¹) 182.0 ± 11.0 174.4 ± 24.2 146.8± 15.3 0.588 0.351 Rate of glucose decline (mg*dl⁻¹*min⁻¹)  2.9 ± 0.4 2.4 ± 0.7  1.1 ± 0.3 0.402 0.117 AUC glucose (0, 60)(mg*dl⁻¹*min⁻¹)10171.4 ± 334.6  11135.6 ± 704.4  10905.9 ± 624.1  0.140 0.811 AUCglucose(0, 180)(mg*dl⁻¹*min⁻¹)  21106 ± 1001.6 27471.6 ± 1963.0 26303.4± 1785.8 0.007 0.667 Subject symptomatic response Overall SymptomScore{circumflex over ( )}  26 ± 3.3 14.6 ± 4.4  4.5 ± 2.2 0.006 0.057Symptom Rise Score+  11 ± 2.4 12.3 ± 3.7  4.3 ± 2.2 0.794 0.087 SymptomFall Score*  22 ± 3.5  4.0 ± 1.5  1.1 ± 0.4 0.001 0.091 ^(a)Data arepresented as mean ± SEM ^(b-c)P-value by two-sided paired student'st-test ^(b)P-value SC Ex(9-39) vs. Placebo ^(c)P-value SC Ex(9-39) vs.IV Ex(9-39)

TABLE 4 37500 75000 125000 pmol/kg pmol/kg pmol/kg n = 1 n = 2 n = 1Injectate Characteristics Fold-increase in dose relative to 5x 10x 15x7,500 pmol/kg Concentration (mg/ml) 15.714 23.57 41.43 Total doseadministered (mg) 11 17 29 Volume per injection (ml) 0.7 0.7 0.7 Numberof injections 1 1 1 Subject Pharmacodynamic Response Fasting plasmaglucose (mg*dl⁻¹) 94 95.8 103.5 Peak postprandial glucose (mg*dl⁻¹) 244226.8 311.0 Time to peak glucose (min) 60 60 60 Nadir postprandialglucose (mg*dl⁻¹) 88 71.3 58.0 Delta peak-nadir glucose (mg*dl⁻¹) 156155.5 253.0 Rate of glucose decline 1 2.2 6.7 (mg*dl⁻¹*min⁻¹) AUCglucose (0, 60) 10815 9694 12713 (mg*dl⁻¹*min⁻¹) AUC glucose(0, 180)28482 25151 25718 (mg*dl⁻¹*min⁻¹) Subject Pharmacokinetic Response Cmax(ng/ml) 114 73.35 56 DN Cmax (ng/mL/mg) 10.36 4.45 1.93 Tmax (h) 3.256.25 5.00 AUCINF [h*ng/ml] 1097 1210 728 DN_AUC_(INF) [h*ng/ml/mg] 69 6324 AUC_(last) (h*ng/ml] 1084 696 720 T^(1/2)(h) 3.60 9.14 3.64MRT_(last) [h]) 6.16 5.74 6.62 MRT_(INF) [h] 6.45 14.66 6.87

TABLE 5 37500 75000 112500 pmol/kg pmol/kg pmol/kg n = 1 n = 1 n = 2Injectate Characteristics Fold-increase in dose relative to 7,500pmol/kg Concentration (mg/ml) 15.714 14.71 13.48 Total dose administered(mg) 11 21 29 Volume per injection (ml) 0.7 0.7 0.7 Number of injections1 2 3 Subject Pharmacodynamic Response Fasting plasma glucose (mg*dl⁻¹)94 90 90.0 Peak postprandial glucose (mg*dl⁻¹) 244 187.0 228.0 Time topeak glucose (min) 60 30 45 Nadir postprandial glucose (mg*dl⁻¹) 88 75.585.0 Delta peak-nadir glucose (mg*dl⁻¹) 156 111.5 143.0 Rate of glucosedecline 1 0.6 1.5 (mg*dl⁻¹*min⁻¹) AUC glucose (0, 60) 10815 9540 11063(mg*dl⁻¹*min⁻¹) AUC glucose(0, 180) 28482 21023 27274 (mg*dl⁻¹*min⁻¹)Subject Pharmacokinetic Response Cmax (ng/ml) 114 158 229 DN Cmax(ng/mL/mg) 10.36 7.52 7.9 Tmax (h) 3.25 4.50 4.50 AUCINF [h*ng/ml] 10971516 1885 DN_AUC_(INF) [h*ng/ml/mg] 69 101 67 AUC_(last) (h*ng/ml] 1084900 1055 T^(1/2)(h) 3.60 4.59 4.87 MRT_(last) [h]) 6.16 4.39 4.69MRT_(INF) [h] 6.45 8.55 9.24

TABLE 6 Dose 2.5 mg 5 mg 5 mg 10 mg Number of subjects dosed n = 1 n = 1n = 1 n = 1 Injectate Characteristics Dose (mg) 2.5 5 5 10 Concentration(mg/ml) 10 mg/ml 10 mg/ml 10 mg/ml 10 mg/ml Volume per injection (ml)0.25 ml 0.5 ml 0.5 ml 1 ml Number of injections 1 1 1 1 Location ofadministration abdomen abdomen arm arm Subject Pharmacodynamic Response% increase AUC glucose (treatment-baseline) 8.8 13.1 16.3 N/A Plasmaglucose nadir ≤50 mg/dL (Yes/No) Yes Yes Yes Yes Subject PharmacokineticResponse Day 3 Cmax (ng/ml) 53.4 77.2 65 128 DN Cmax (ng/ml/mg) 21.3615.44 13 12.8 Tmax Day 3 (h) 4 5 5 5 AUClast (h*ng/ml] 379.2 409.1 634.3969 T½(h) 4.3 4.4 4.6 6.5

The invention claimed is:
 1. A method for treating hyperinsulinemichypoglycemia in a subject, the method comprising subcutaneouslyadministering to the subject in need thereof an isotonic solutioncomprising a therapeutically effective amount of exendin(9-39) and atleast one of an antimicrobial preservative, a tonicity adjusting agent,or a buffer; wherein the therapeutically effective amount is 2-100 mg ofexendin(9-39); and wherein the exendin(9-39) is administered at aconcentration of 20-45 mg/ml.
 2. The method of claim 1, wherein thetonicity adjusting agent comprises mannitol.
 3. The method of claim 1,wherein the therapeutically effective amount is 5-40 mg ofexendin(9-39).
 4. The method of claim 1, wherein the exendin(9-39) isadministered twice per day.
 5. The method of claim 1, wherein theexendin(9-39) is administered three times per day.
 6. The method ofclaim 1, wherein the patient has previously had bariatric surgery. 7.The method of claim 6, wherein the bariatric surgery is Roux-en-Ygastric bypass, vertical sleeve gastrectomy, placement of an endosleevedevice, duodenal mucosal resurfacing partial bypass of the duodenum,vagal nerve blockade, or pyloroplasty.
 8. The method of claim 1, whereinthe patient has previously had gastrointestinal surgery.
 9. The methodof claim 8, wherein the gastrointestinal surgery is gastrectomy, NissenFundoplication, or esophagectomy.
 10. A method for treatinghyperinsulinemic hypoglycemia in a subject, the method comprisingsubcutaneously administering to the subject in need thereof atherapeutically effective amount of exendin(9-39), wherein thetherapeutically effective amount is 2-100 mg of exendin(9-39); whereinthe exendin(9-39) is encapsulated in an extended release capsule; andwherein the exendin(9-39) is administered at a concentration of 20-45mg/ml.
 11. The method of claim 10, wherein the extended release capsulecomprises a microsphere.
 12. The method of claim 11, wherein themicrosphere comprises a biopolymer.
 13. The method of claim 10, whereinthe extended release capsule is a nano-lipocapsule.
 14. The method ofclaim 10, wherein the therapeutically effective amount is 5-20 mg ofexendin(9-39).
 15. The method of claim 10, wherein the exendin(9-39) isadministered once per day.
 16. The method of claim 10, wherein theexendin(9-39) is administered once per week.
 17. The method of claim 10,wherein the patient has previously had bariatric surgery.
 18. The methodof claim 17, wherein the bariatric surgery is Roux-en-Y gastric bypass,vertical sleeve gastrectomy, placement of an endosleeve device, duodenalmucosal resurfacing partial bypass of the duodenum, vagal nerveblockade, or pyloroplasty.
 19. The method of claim 10, wherein thepatient has previously had gastrointestinal surgery.
 20. The method ofclaim 19, wherein the gastrointestinal surgery is gastrectomy, NissenFundoplication, or esophagectomy.
 21. A method for treatinghyperinsulinemic hypoglycemia in a subject, the method comprising:solubilizing lyophilized exendin(9-39) with saline or otherpharmacologically acceptable diluent to form an exendin(9-39) solution;and subcutaneously administering to the subject in need thereof atherapeutically effective amount of the exendin(9-39) solution; whereinthe therapeutically effective amount is 2-100 mg of exendin(9-39); andwherein the exendin(9-39) is administered at a concentration of 20-45mg/ml.
 22. The method of claim 21, wherein the lyophilized exendin(9-39)is provided in dual-chamber cartridge.
 23. The method of claim 21,wherein the exendin(9-39) solution is administered using a pen injector.24. A method for treating hyperinsulinemic hypoglycemia in a subject,the method comprising: subcutaneously administering to the subject inneed thereof a therapeutically effective amount of exendin(9-39) for afirst treatment period, wherein the exendin(9-39) is administered at afirst dosage; and subcutaneously administering to the subject atherapeutically effective amount of exendin(9-39) for a second treatmentperiod after the first treatment period, wherein the exendin(9-39) isadministered at a second dosage; wherein the exendin(9-39) isadministered at a concentration of 20-45 mg/ml.
 25. The method of claim24, wherein the second dosage is greater than the first dosage.
 26. Themethod of claim 24, further comprising subcutaneously administering tothe subject a therapeutically effective amount of exendin(9-39) for athird treatment period after the second treatment period, wherein theexendin(9-39) is administered at a third dosage.
 27. The method of claim26, wherein the third dosage is greater than the second dosage.
 28. Themethod of claim 24, wherein the first dosage is from 5-10 mg ofexendin(9-39).
 29. The method of claim 24, wherein the second dosage isfrom 10-20 mg of exendin(9-39).
 30. The method of claim 26, wherein thethird dosage is from 20-30 mg of exendin(9-39).
 31. A method fortreating hyperinsulinemic hypoglycemia in a subject, the methodcomprising administering to the subject in need thereof atherapeutically effective amount of exendin(9-39) once per day or twiceper day, wherein the therapeutically effective amount is 2-100 mg ofexendin(9-39); wherein the exendin(9-39) is administered at aconcentration of 20-45 mg/ml; and wherein the exendin(9-39) isadministered parenterally, transmucosally, transdermally,intramuscularly, intravenously, intradermally, intra-peritonealy,orally, nasally, or by inhalation.
 32. The method of claim 31, whereinthe exendin(9-39) is administered orally, and wherein the exendin(9-39)is co-administered with at least one of a protease inhibitor or anabsorption enhancer.